Xylanases, nucleic acids encoding them and methods for making and using them

ABSTRACT

The invention relates to xylanases and to polynucleotides encoding the xylanases. In addition, methods of designing new xylanases and methods of use thereof are also provided. The xylanases have increased activity and stability at increased pH and temperature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. Provisional Application No. 60/389,299, filed Jun. 14, 2002. Theaforementioned application is explicitly incorporated herein byreference in its entirety and for all purposes.

FIELD OF THE INVENTION

This invention relates generally to enzymes, polynucleotides encodingthe enzymes, the use of such polynucleotides and polypeptides and morespecifically to enzymes having xylanase activity, e.g., catalyzinghydrolysis of internal β-1,4-xylosidic linkages or endo-β-1,4-glucanaselinkages.

BACKGROUND

Xylanases (e.g., endo-1,4-beta-xylanase, EC 3.2.1.8) hydrolyze internalβ-1,4-xylosidic linkages in xylan to produce smaller molecular weightxylose and xylo-oligomers. Xylans are polysaccharides formed from1,4-β-glycoside-linked D-xylopyranoses. Xylanases are of considerablecommercial value, being used in the food industry, for baking and fruitand vegetable processing, breakdown of agricultural waste, in themanufacture of animal feed and in pulp and paper production. Xylanasesare formed by fungi and bacteria.

Arabinoxylanase are major non-starch polysaccharides of cerealsrepresenting 2.5-7.1% w/w depending on variety and growth conditions.The physicochemical properties of this polysaccharide are such that itgives rise to viscous solutions or even gels under oxidative conditions.In addition, arabinoxylans have high water-binding capacity and may havea role in protein foam stability. All of these characteristics presentproblems for several industries including brewing, baking, animalnutrition and paper manufacturing. In brewing applications, the presenceof xylan results in wort filterability and haze formation issues. Inbaking applications (especially for cookies and crackers), thesearabinoxylans create sticky doughs that are difficult to machine andreduce biscuit size. In addition, this carbohydrate is implicated inrapid rehydration of the baked product resulting in loss of crispinessand reduced shelf-life. For monogastric animal feed applications withcereal diets, arabinoxylan is a major contributing factor to viscosityof gut contents and thereby adversely affects the digestibility of thefeed and animal growth rate. For ruminant animals, these polysaccharidesrepresent substantial components of fiber intake and more completedigestion of arabinoxylans would facilitate higher feed conversionefficiencies.

Xylanases are currently used as additives (dough conditioners) in doughprocessing for the hydrolysis of water soluble arabinoxylan. In bakingapplications (especially for cookies and crackers), arabinoxylan createssticky doughs that are difficult to machine and reduce biscuit size. Inaddition, this carbohydrate is implicated in rapid rehydration of thebaked product resulting in loss of crispiness and reduced shelf-life.

The enhancement of xylan digestion in animal feed may improve theavailability and digestibility of valuable carbohydrate and protein feednutrients. For monogastric animal feed applications with cereal diets,arabinoxylan is a major contributing factor to viscosity of gut contentsand thereby adversely affects the digestibility of the feed and animalgrowth rate. For ruminant animals, these polysaccharides representsubstantial components of fiber intake and more complete digestion wouldfacilitate higher feed conversion efficiencies. It is desirable foranimal feed xylanases to be active in the animal stomach. This requiresa feed enzyme to have high activity at 37° C. and at low pH formonogastrics (pH 2-4) and near neutral pH for ruminants (pH 6.5-7). Theenzyme should also possess resistance to animal gut xylanases andstability at the higher temperatures involved in feed pelleting. Assuch, there is a need in the art for xylanase feed additives formonogastric feed with high specific activity, activity at 35-40° C. andpH 2-4, half life greater than 30 minutes in SGF and a half-life >5minutes at 85° C. in formulated state. For ruminant feed, there is aneed for xylanase feed additives that have a high specific activity,activity at 35-40° C. and pH 6.5-7.0, half life greater than 30 minutesin SRF and stability as a concentrated dry powder.

Xylanases are also used in a number of other applications. For example,xylanases are used in improving the quality and quantity of milk proteinproduction in lactating cows (see, for example, Kung, L., et al, J.Dairy Science, 2000 Jan 83:115-122), increasing the amount of solublesaccharides in the stomach and small intestine of pigs (see, forexample, van der Meulen, J. et al, Arch. Tieremahr, 2001 54:101-115),improving late egg production efficiency and egg yields in hens (see,for example, Jaroni, D., et al, Poult. Sci., 1999 June 78:841-847).Additionally, xylanases have been shown to be useful in biobleaching andtreatment of chemical pulps (see, for example, U.S. Pat. No. 5,202,249),biobleaching and treatment of wood or paper pulps (see, for example,U.S. Pat. Nos. 5,179,021, 5,116,746, 5,407,827, 5,405,769, 5,395,765,5,369,024, 5,457,045, 5,434,071, 5,498,534, 5,591,304, 5,645,686,5,725,732, 5,759,840, 5,834,301, 5,871,730 and 6,057,438) in reducinglignin in wood and modifying wood (see, for example, U.S. Pat. Nos.5,486,468 and 5,770,012) as flour, dough and bread improvers (see, forexample, U.S. Pat. Nos. 5,108,765 and 5,306,633) as feed additivesand/or supplements, as set forth above (see, for example, U.S. Pat. Nos.5,432,074, 5,429,828, 5,612,055, 5,720,971, 5,981,233, 5,948,667,6,099,844, 6,132,727 and 6,132,716), in manufacturing cellulosesolutions (see, for example, U.S. Pat. No. 5,760,211). Detergentcompositions having xylanase activity are used for fruit, vegetablesand/or mud and clay compounds (see, for example, U.S. Pat. No.5,786,316).

Xylanases are also useful in a method of use and composition of acarbohydrase and/or a xylanase for the manufacture of an agent for thetreatments and/or prophylaxis of coccidiosis. The manufactured agent canbe in the form of a cereal-based animal feed. (see, for example, U.S.Pat. No. 5,624,678) Additional uses for xylanases include use in theproduction of water soluble dietary fiber (see, for example, U.S. Pat.No. 5,622,738), in improving the filterability, separation andproduction of starch (see, for example, U.S. Pat. Nos. 4,960,705 and5,023,176), in the beverage industry in improving filterability of wortor beer (see, for example, U.S. Pat. No. 4,746,517), in an enzymecomposition for promoting the secretion of milk of livestock andimproving the quality of the milk (see, for example, U.S. Pat. No.4,144,354), in reducing viscosity of plant material (see, for example,U.S. Pat. No. 5,874,274), in increasing viscosity or gel strength offood products such as jam, marmalade, jelly, juice, paste, soup, salsa,etc. (see, for example, U.S. Pat. No. 6,036,981). Xylanases may also beused in hydrolysis of hemicellulose for which it is selective,particularly in the presence of cellulose. Additionally, the cellulaserich retentate is suitable for the hydrolysis of cellulose (see, forexample, U.S. Pat. No. 4,725,544).

Various uses of xylanases include the production of ethanol (see, forexample, PCT Application Nos. WO0043496 and WO8100857), intransformation of a microbe that produces ethanol (see, for example, PCTApplication No. WO99/46362), in production of oenological tannins andenzymatic composition (see, for example, PCT Application No. WO0164830),in stimulating the natural defenses of plants (see, for example, PCTApplication No. WO0130161), in production of sugars from hemicellulosesubstrates (see, for example, PCT Application No. WO9203541), in thecleaning of fruit, vegetables, mud or clay containing soils (see, forexample, PCT Application No. WO9613568), in cleaning beer filtrationmembranes (see, for example, PCT Application No. WO9623579), in a methodof killing or inhibiting microbial cells (see, for example, PCTApplication No. WO9732480) and in determining the characteristics ofprocess waters from wood pulp bleaching by using the ratios of two UVabsorption measurements and comparing the spectra (see, for example, PCTApplication No. WO9840721).

With regard to xylanases used in the paper and pulp industry, xylanaseshave been isolated from many sources. In particular, see U.S. Pat. Nos.6,083,733 and 6,140,095 and 6,346,407. In particular, it is noted thatU.S. Pat. No. 6,140,095 addresses alkali-tolerant xylanases. However, itis noted that there remains a need in the art for xylanases to be usedin the paper and pulp industry where the enzyme is active in thetemperature range of 65° C. to 75° C. and at a pH of approximately 10.Additionally, an enzyme of the invention useful in the paper and pulpindustry would decrease the need for bleaching chemicals, such aschlorine dioxide.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention, e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ IDNO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,SEQ ID NO:377 or SEQ ID NO:379, over a region of at least about 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350,2400, 2450, 2500, or more residues, encodes at least one polypeptidehaving a xylanase activity, and the sequence identities are determinedby analysis with a sequence comparison algorithm or by a visualinspection.

Exemplary nucleic acids of the invention also include isolated orrecombinant nucleic acids encoding a polypeptide having a sequence asset forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:10, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ IDNO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166,SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ IDNO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194,SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ IDNO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222,SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ IDNO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250,SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ IDNO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278,SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ IDNO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306,SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ IDNO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334,SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ IDNO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362,SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ IDNO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378 or SEQ ID NO:380,and subsequences thereof and variants thereof. In one aspect, thepolypeptide has a xylanase activity.

In one aspect, the invention also provides xylanase-encoding nucleicacids with a common novelty in that they are derived from mixedcultures. The invention provides xylanase-encoding nucleic acidsisolated from mixed cultures comprising a nucleic acid sequence havingat least about 10, 15, 20, 25, 30, 35, 40, 45, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention, e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ 10 NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ IDNO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,SEQ ID NO:377 or SEQ ID NO:379, over a region of at least about 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, or more.

In one aspect, the invention also provides xylanase-encoding nucleicacids with a common novelty in that they are derived from anenvironmental source, e.g., mixed environmental sources, a bacterialsource and/or an archaeal source, see Table 3, below. In one aspect, theinvention provides xylanase-encoding nucleic acids isolated from anenvironmental source, e.g., a mixed environmental source, a bacterialsource and/or an archaeal source, comprising a nucleic acid sequencehaving at least about 10, 15, 20, 25, 30, 35, 40, 45, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary nucleic acid of the invention over a region of at leastabout 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more,residues, wherein the nucleic acid encodes at least one polypeptidehaving a xylanase activity, and the sequence identities are determinedby analysis with a sequence comparison algorithm or by a visualinspection.

In one aspect, the invention also provides xylanase-encoding nucleicacids with a common novelty in that they are derived from a commonglycosidase family, e.g., family 5, 6, 8, 10, 11, 26 or 30, as set forthin Table 5, below.

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall -p blastp-d “nr pataa”-F F, and all other options are set to default.

Another aspect of the invention is an isolated or recombinant nucleicacid including at least 10 consecutive bases of a nucleic acid sequenceof the invention, sequences substantially identical thereto, and thesequences complementary thereto.

In one aspect, the xylanase activity comprises catalyzing hydrolysis ofinternal β-1,4-xylosidic linkages. In one aspect, the xylanase activitycomprises an endo-1,4-beta-xylanase activity.

In one aspect, the xylanase activity comprises hydrolyzing a xylan toproduce a smaller molecular weight xylose and xylo-oligomer. In oneaspect, the xylan comprises an arabinoxylan, such as a water solublearabinoxylan. The water soluble arabinoxylan can comprise a dough or abread product.

In one aspect, the xylanase activity comprises hydrolyzingpolysaccharides comprising 1,4-β-glycoside-linked D-xylopyranoses. Inone aspect, the xylanase activity comprises hydrolyzing hemicelluloses.In one aspect, the xylanase activity comprises hydrolyzinghemicelluloses in a wood or paper pulp or a paper product. In oneaspect, the invention provides methods for reducing lignin in a wood orwood product comprising contacting the wood or wood product with apolypeptide of the invention.

In one aspect, the xylanase activity comprises catalyzing hydrolysis ofxylans in a beverage or a feed or a food product. The feed or foodproduct can comprise a cereal-based animal feed, a wort or a beer, amilk or a milk product, a fruit or a vegetable. In one aspect, theinvention provides a food, feed or beverage or a beverage precursorcomprising a polypeptide of the invention. The food can be a dough or abread product. The beverage or a beverage precursor can be a beer or awort.

In one aspect, the invention provides methods of dough conditioningcomprising contacting a dough or a bread product with at least onepolypeptide of the invention under conditions sufficient forconditioning the dough. In one aspect, the invention provides methods ofbeverage production comprising administration of at least onepolypeptide of the invention to a beverage or a beverage precursor underconditions sufficient for decreasing the viscosity of the beverage.

In one aspect, the xylanase activity comprises catalyzing hydrolysis ofxylans in a cell, e.g., a plant cell or a microbial cell.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a xylanase activity that is thermostable. Thepolypeptide can retain a xylanase activity under conditions comprising atemperature range of between about 37° C. to about 95° C.; between about55° C. to about 85° C., between about 70° C. to about 95° C., or,between about 90° C. to about 95° C.

In another aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a xylanase activity that is thermotolerant. Thepolypeptide can retain a xylanase activity after exposure to atemperature in the range from greater than 37° C. to about 95° C. oranywhere in the range from greater than 55° C. to about 85° C. Thepolypeptide can retain a xylanase activity after exposure to atemperature in the range between about 1° C. to about 5° C., betweenabout 5° C. to about 15° C., between about 15° C. to about 25° C.,between about 25° C. to about 37° C., between about 37° C. to about 95°C., between about 55° C. to about 85° C., between about 70° C. to about75° C., or between about 90° C. to about 95° C., or more. In one aspect,the polypeptide retains a xylanase activity after exposure to atemperature in the range from greater than 90° C. to about 95° C. at pH4.5.

The invention provides isolated or recombinant nucleic acids comprisinga sequence that hybridizes under stringent conditions to a nucleic acidcomprising a sequence of the invention, e.g., a sequence as set forth inSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:157, SEQ ID NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ IDNO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203,SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ IDNO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231,SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ IDNO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259,SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ IDNO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287,SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315,SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ IDNO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343,SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ IDNO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371,SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377 or SEQ ID NO:379, orfragments or subsequences thereof. In one aspect, the nucleic acidencodes a polypeptide having a xylanase activity. The nucleic acid canbe at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200 or more residues in length or the fulllength of the gene or transcript. In one aspect, the stringentconditions include a wash step comprising a wash in 0.2×SSC at atemperature of about 65° C. for about 15 minutes.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having a xylanase activity, wherein theprobe comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more,consecutive bases of a sequence comprising a sequence of the invention,or fragments or subsequences thereof, wherein the probe identifies thenucleic acid by binding or hybridization. The probe can comprise anoligonucleotide comprising at least about 10 to 50, about 20 to 60,about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases ofa sequence comprising a sequence of the invention, or fragments orsubsequences thereof.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having a xylanase activity, wherein theprobe comprises a nucleic acid comprising a sequence at least about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or moreresidues having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to a nucleic acid of theinvention, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by visual inspection.

The probe can comprise an oligonucleotide comprising at least about 10to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to100 consecutive bases of a nucleic acid sequence of the invention, or asubsequence thereof.

The invention provides an amplification primer pair for amplifying anucleic acid encoding a polypeptide having a xylanase activity, whereinthe primer pair is capable of amplifying a nucleic acid comprising asequence of the invention, or fragments or subsequences thereof. One oreach member of the amplification primer sequence pair can comprise anoligonucleotide comprising at least about 10 to 50 consecutive bases ofthe sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of a nucleic acid of theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more residues of the complementary strand ofthe first member.

The invention provides xylanase-encoding nucleic acids generated byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. The invention providesxylanases generated by amplification, e.g., polymerase chain reaction(PCR), using an amplification primer pair of the invention. Theinvention provides methods of making a xylanase by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. In one aspect, the amplification primer pair amplifies anucleic acid from a library, e.g., a gene library, such as anenvironmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having a xylanase activity comprising amplification of atemplate nucleic acid with an amplification primer sequence pair capableof amplifying a nucleic acid sequence of the invention, or fragments orsubsequences thereof.

The invention provides expression cassettes comprising a nucleic acid ofthe invention or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a cereal, a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a cereal plant, a corn plant, apotato plant, a tomato plant, a wheat plant, an oilseed plant, arapeseed plant, a soybean plant, a rice plant, a barley plant or atobacco plant.

The invention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be a cereal plant, a corn seed, a wheat kernel,an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed,a sesame seed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of a xylanase message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention. In one aspect, the antisenseoligonucleotide is between about 10 to 50, about 20 to 60, about 30 to70, about 40 to 80, or about 60 to 100 bases in length.

The invention provides methods of inhibiting the translation of axylanase message in a cell comprising administering to the cell orexpressing in the cell an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesdouble-stranded inhibitory RNA (RNAi) molecules comprising a subsequenceof a sequence of the invention. In one aspect, the RNAi is about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.The invention provides methods of inhibiting the expression of axylanase in a cell comprising administering to the cell or expressing inthe cell a double-stranded inhibitory RNA (iRNA), wherein the RNAcomprises a subsequence of a sequence of the invention.

The invention provides an isolated or recombinant polypeptide comprisingan amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary polypeptide or peptide of the invention over a region of atleast about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350 or more residues, or over the full length of the polypeptide,and the sequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection. Exemplary polypeptide orpeptide sequences of the invention include SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ IDNO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ IDNO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ IDNO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ IDNO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ IDNO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ IDNO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ IDNO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340,SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ IDNO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368,SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ IDNO:378 or SEQ ID NO:380, and subsequences thereof and variants thereof.Exemplary polypeptides also include fragments of at least about 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600 or more residues in length, or over the full length of anenzyme. Exemplary polypeptide or peptide sequences of the inventioninclude sequence encoded by a nucleic acid of the invention. Exemplarypolypeptide or peptide sequences of the invention include polypeptidesor peptides specifically bound by an antibody of the invention. In oneaspect, a polypeptide of the invention has at least one xylanaseactivity.

Another aspect of the invention provides an isolated or recombinantpolypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutivebases of a polypeptide or peptide sequence of the invention, sequencessubstantially identical thereto, and the sequences complementarythereto. The peptide can be, e.g., an immunogenic fragment, a motif(e.g., a binding site), a signal sequence, a prepro sequence or anactive site.

The invention provides isolated or recombinant nucleic acids comprisinga sequence encoding a polypeptide having a xylanase activity and asignal sequence, wherein the nucleic acid comprises a sequence of theinvention. The signal sequence can be derived from another xylanase or anon-xylanase (a heterologous) enzyme. The invention provides isolated orrecombinant nucleic acids comprising a sequence encoding a polypeptidehaving a xylanase activity, wherein the sequence does not contain asignal sequence and the nucleic acid comprises a sequence of theinvention.

In one aspect, the xylanase activity comprises catalyzing hydrolysis ofinternal β-1,4-xylosidic linkages. In one aspect, the xylanase activitycomprises an endo-1,4-beta-xylanase activity. In one aspect, thexylanase activity comprises hydrolyzing a xylan to produce a smallermolecular weight xylose and xylo-oligomer. In one aspect, the xylancomprises an arabinoxylan, such as a water soluble arabinoxylan. Thewater soluble arabinoxylan can comprise a dough or a bread product.

In one aspect, the xylanase activity comprises hydrolyzingpolysaccharides comprising 1,4-β-glycoside-linked D-xylopyranoses. Inone aspect, the xylanase activity comprises hydrolyzing hemicelluloses.In one aspect, the xylanase activity comprises hydrolyzinghemicelluloses in a wood or paper pulp or a paper product.

In one aspect, the xylanase activity comprises catalyzing hydrolysis ofxylans in a feed or a food product. The feed or food product cancomprise a cereal-based animal feed, a wort or a beer, a milk or a milkproduct, a fruit or a vegetable.

In one aspect, the xylanase activity comprises catalyzing hydrolysis ofxylans in a cell, e.g., a plant cell or a microbial cell.

In one aspect, the xylanase activity is thermostable. The polypeptidecan retain a xylanase activity under conditions comprising a temperaturerange of between about 1° C. to about 5° C., between about 5° C. toabout 15° C., between about 15° C. to about 25° C., between about 25° C.to about 37° C., between about 37° C. to about 95° C., between about 55°C. to about 85° C., between about 70° C. to about 75° C., or betweenabout 90° C. to about 95° C., or more. In another aspect, the xylanaseactivity can be thermotolerant. The polypeptide can retain a xylanaseactivity after exposure to a temperature in the range from greater than37° C. to about 95° C., or in the range from greater than 55° C. toabout 85° C. In one aspect, the polypeptide can retain a xylanaseactivity after exposure to a temperature in the range from greater than90° C. to about 95° C. at pH 4.5.

In one aspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention that lacks a signal sequence. In oneaspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention comprising a heterologous signal sequence,such as a heterologous xylanase or non-xylanase signal sequence.

In one aspect, the invention provides chimeric proteins comprising afirst domain comprising a signal sequence of the invention and at leasta second domain. The protein can be a fusion protein. The second domaincan comprise an enzyme. The enzyme can be a xylanase.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP), a prepro sequence and/or acatalytic domain (CD) of the invention and at least a second domaincomprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro sequence and/or catalytic domain (CD). Inone aspect, the heterologous polypeptide or peptide is not a xylanase.The heterologous polypeptide or peptide can be amino terminal to,carboxy terminal to or on both ends of the signal peptide (SP), preprosequence and/or catalytic domain (CD).

The invention provides isolated or recombinant nucleic acids encoding achimeric polypeptide, wherein the chimeric polypeptide comprises atleast a first domain comprising signal peptide (SP), a prepro domainand/or a catalytic domain (CD) of the invention and at least a seconddomain comprising a heterologous polypeptide or peptide, wherein theheterologous polypeptide or peptide is not naturally associated with thesignal peptide (SP), prepro domain and/or catalytic domain (CD).

The invention provides isolated or recombinant signal sequences (e.g.,signal peptides) consisting of a sequence as set forth in residues 1 to14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to38, 1 to 40, 1 to 41, 1 to 42, 1 to 43 or 1 to 44, of a polypeptide ofthe invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58,SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78,SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88,SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ IDNO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126,SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:134; SEQ IDNO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144;NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164,SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ IDNO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192,SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ IDNO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220,SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ IDNO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248,SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ IDNO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276,SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ IDNO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304,SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ IDNO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332,SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ IDNO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360,SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ IDNO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378 orSEQ ID NO:380.

In one aspect, the xylanase activity comprises a specific activity atabout 37° C. in the range from about 1 to about 1200 units per milligramof protein, or, about 100 to about 1000 units per milligram of protein.In another aspect, the xylanase activity comprises a specific activityfrom about 100 to about 1000 units per milligram of protein, or, fromabout 500 to about 750 units per milligram of protein. Alternatively,the xylanase activity comprises a specific activity at 37° C. in therange from about 1 to about 750 units per milligram of protein, or, fromabout 500 to about 1200 units per milligram of protein. In one aspect,the xylanase activity comprises a specific activity at 37° C. in therange from about 1 to about 500 units per milligram of protein, or, fromabout 750 to about 1000 units per milligram of protein. In anotheraspect, the xylanase activity comprises a specific activity at 37° C. inthe range from about 1 to about 250 units per milligram of protein.Alternatively, the xylanase activity comprises a specific activity at37° C. in the range from about 1 to about 100 units per milligram ofprotein. In another aspect, the thermotolerance comprises retention ofat least half of the specific activity of the xylanase at 37° C. afterbeing heated to the elevated temperature. Alternatively, thethermotolerance can comprise retention of specific activity at 37° C. inthe range from about 1 to about 1200 units per milligram of protein, or,from about 500 to about 1000 units per milligram of protein, after beingheated to the elevated temperature. In another aspect, thethermotolerance can comprise retention of specific activity at 37° C. inthe range from about 1 to about 500 units per milligram of protein afterbeing heated to the elevated temperature.

The invention provides the isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

In one aspect, the polypeptide can retain a xylanase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.In another aspect, the polypeptide can retain a xylanase activity underconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5 or pH 1. In one aspect, the polypeptide can retain axylanase activity after exposure to conditions comprising about pH 6.5,pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptidecan retain a xylanase activity after exposure to conditions comprisingabout pH 7, pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH11.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second protein or domain. The second member of theheterodimer can be a different phospholipase, a different enzyme oranother protein. In one aspect, the second domain can be a polypeptideand the heterodimer can be a fusion protein. In one aspect, the seconddomain can be an epitope or a tag. In one aspect, the invention provideshomodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having a xylanaseactivity, wherein the polypeptide comprises a polypeptide of theinvention, a polypeptide encoded by a nucleic acid of the invention, ora polypeptide comprising a polypeptide of the invention and a seconddomain. In one aspect, the polypeptide can be immobilized on a cell, ametal, a resin, a polymer, a ceramic, a glass, a microelectrode, agraphitic particle, a bead, a gel, a plate, an array or a capillarytube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention, e.g., an antibody thatspecifically binds to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention.

The invention provides method of isolating or identifying a polypeptidehaving a xylanase activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying apolypeptide having a xylanase activity.

The invention provides methods of making an anti-xylanase antibodycomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate a humoral immune response, therebymaking an anti-xylanase antibody. The invention provides methods ofmaking an anti-xylanase immune comprising administering to a non-humananimal a nucleic acid of the invention or a polypeptide of the inventionor subsequences thereof in an amount sufficient to generate an immuneresponse.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide having axylanase activity comprising the following steps: (a) providing apolypeptide of the invention; or a polypeptide encoded by a nucleic acidof the invention; (b) providing a xylanase substrate; and (c) contactingthe polypeptide or a fragment or variant thereof of step (a) with thesubstrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of a reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product detects a polypeptide having a xylanase activity.

The invention provides methods for identifying a xylanase substratecomprising the following steps: (a) providing a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of a reaction product identifies the testsubstrate as a xylanase substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of a xylanaseactivity comprising the following steps: (a) providing a polypeptide ofthe invention or a polypeptide encoded by a nucleic acid of theinvention; (b) providing a test compound; (c) contacting the polypeptideof step (a) with the test compound of step (b) and measuring an activityof the xylanase, wherein a change in the xylanase activity measured inthe presence of the test compound compared to the activity in theabsence of the test compound provides a determination that the testcompound modulates the xylanase activity. In one aspect, the xylanaseactivity can be measured by providing a xylanase substrate and detectinga decrease in the amount of the substrate or an increase in the amountof a reaction product, or, an increase in the amount of the substrate ora decrease in the amount of a reaction product. A decrease in the amountof the substrate or an increase in the amount of the reaction productwith the test compound as compared to the amount of substrate orreaction product without the test compound identifies the test compoundas an activator of xylanase activity. An increase in the amount of thesubstrate or a decrease in the amount of the reaction product with thetest compound as compared to the amount of substrate or reaction productwithout the test compound identifies the test compound as an inhibitorof xylanase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention(e.g., a polypeptide encoded by a nucleic acid of the invention). In oneaspect, the computer system can further comprise a sequence comparisonalgorithm and a data storage device having at least one referencesequence stored thereon. In another aspect, the sequence comparisonalgorithm comprises a computer program that indicates polymorphisms. Inone aspect, the computer system can further comprise an identifier thatidentifies one or more features in said sequence. The invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence of the invention. The invention provides methodsfor identifying a feature in a sequence comprising the steps of: (a)reading the sequence using a computer program which identifies one ormore features in a sequence, wherein the sequence comprises apolypeptide sequence or a nucleic acid sequence of the invention; and(b) identifying one or more features in the sequence with the computerprogram. The invention provides methods for comparing a first sequenceto a second sequence comprising the steps of: (a) reading the firstsequence and the second sequence through use of a computer program whichcompares sequences, wherein the first sequence comprises a polypeptidesequence or a nucleic acid sequence of the invention; and (b)determining differences between the first sequence and the secondsequence with the computer program. The step of determining differencesbetween the first sequence and the second sequence can further comprisethe step of identifying polymorphisms. In one aspect, the method canfurther comprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a xylanase activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide having a xylanase activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to the amplification primer pair; and, (c) combiningthe nucleic acid of step (b) with the amplification primer pair of step(a) and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide having axylanase activity from an environmental sample. One or each member ofthe amplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of a sequence ofthe invention. In one aspect, the amplification primer sequence pair isan amplification pair of the invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a xylanase activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid of the invention or asubsequence thereof; (b) isolating a nucleic acid from the environmentalsample or treating the environmental sample such that nucleic acid inthe sample is accessible for hybridization to a polynucleotide probe ofstep (a); (c) combining the isolated nucleic acid or the treatedenvironmental sample of step (b) with the polynucleotide probe of step(a); and (d) isolating a nucleic acid that specifically hybridizes withthe polynucleotide probe of step (a), thereby isolating or recovering anucleic acid encoding a polypeptide having a xylanase activity from anenvironmental sample. The environmental sample can comprise a watersample, a liquid sample, a soil sample, an air sample or a biologicalsample. In one aspect, the biological sample can be derived from abacterial cell, a protozoan cell, an insect cell, a yeast cell, a plantcell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having a xylanase activity comprising the stepsof: (a) providing a template nucleic acid comprising a nucleic acid ofthe invention; and (b) modifying, deleting or adding one or morenucleotides in the template sequence, or a combination thereof, togenerate a variant of the template nucleic acid. In one aspect, themethod can further comprise expressing the variant nucleic acid togenerate a variant xylanase polypeptide. The modifications, additions ordeletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (e.g., GeneReassembly™, see, e.g., U.S.Pat. No. 6,537,776), gene site saturated mutagenesis (GSSM™), syntheticligation reassembly (SLR) or a combination thereof. In another aspect,the modifications, additions or deletions are introduced by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until a xylanasehaving an altered or different activity or an altered or differentstability from that of a polypeptide encoded by the template nucleicacid is produced. In one aspect, the variant xylanase polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature. In another aspect, the variant xylanasepolypeptide has increased glycosylation as compared to the xylanaseencoded by a template nucleic acid. Alternatively, the variant xylanasepolypeptide has a xylanase activity under a high temperature, whereinthe xylanase encoded by the template nucleic acid is not active underthe high temperature. In one aspect, the method can be iterativelyrepeated until a xylanase coding sequence having an altered codon usagefrom that of the template nucleic acid is produced. In another aspect,the method can be iteratively repeated until a xylanase gene havinghigher or lower level of message expression or stability from that ofthe template nucleic acid is produced.

In one aspect, the invention provides isolated or recombinant nucleicacids comprising a sequence as set forth in SEQ ID NO:189, wherein SEQID NO:189 contains one or more of the following mutations: thenucleotides at positions 22 to 24 are TTC, the nucleotides at positions31 to 33 are CAC, the nucleotides at positions 34 to 36 are TTG, thenucleotides at positions 49 to 51 are ATA, the nucleotides at positions31 to 33 are CAT, the nucleotides at positions 67 to 69 are ACG, thenucleotides at positions 178 to 180 are CAC, the nucleotides atpositions 190 to 192 are TGT, the nucleotides at positions 190 to 192are GTA, the nucleotides at positions 190 to 192 are GTT, thenucleotides at positions 193 to 195 are GTG, the nucleotides atpositions 202 to 204 are GCT, the nucleotides at positions 235 to 237are CCA, or the nucleotides at positions 235 to 237 are CCC. In oneaspect, the invention provides methods for making a nucleic acidcomprising this sequence, wherein the mutations in SEQ ID NO:189 areobtained by gene site saturated mutagenesis (GSSM™).

In one aspect, the invention provides isolated or recombinant nucleicacids comprising SEQ ID NO:190, wherein SEQ ID NO:190 contains one ormore of the following mutations: the aspartic acid at amino acidposition 8 is phenylalanine, the glutamine at amino acid position 11 ishistidine, the asparagine at amino acid position 12 is leucine, theglycine at amino acid position 17 is isoleucine, the threonine at aminoacid position 23 is threonine encoded by a codon other than the wildtype codon, the glycine at amino acid position 60 is histidine, theproline at amino acid position 64 is cysteine, the proline at amino acidposition 64 is valine, the serine at amino acid position 65 is valine,the glycine at amino acid position 68 is isoleucine, the glycine atamino acid position 68 is alanine, or the valine at amino acid position79 is proline.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a xylanase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a polypeptidehaving a xylanase activity; and, (b) identifying a non-preferred or aless preferred codon in the nucleic acid of step (a) and replacing itwith a preferred or neutrally used codon encoding the same amino acid asthe replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in the host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a xylanase activity; the method comprisingthe following steps: (a) providing a nucleic acid of the invention; and,(b) identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding a xylanase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a xylanase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a xylanasepolypeptide; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having a xylanase activity to decrease itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention; and (b) identifying atleast one preferred codon in the nucleic acid of step (a) and replacingit with a non-preferred or less preferred codon encoding the same aminoacid as the replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in a host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified xylanase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising the following steps: (a) providing a first nucleicacid encoding a first active site or first substrate binding site,wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a nucleic acid of theinvention, and the nucleic acid encodes a xylanase active site or axylanase substrate binding site; (b) providing a set of mutagenicoligonucleotides that encode naturally-occurring amino acid variants ata plurality of targeted codons in the first nucleic acid; and, (c) usingthe set of mutagenic oligonucleotides to generate a set of activesite-encoding or substrate binding site-encoding variant nucleic acidsencoding a range of amino acid variations at each amino acid codon thatwas mutagenized, thereby producing a library of nucleic acids encoding aplurality of modified xylanase active sites or substrate binding sites.In one aspect, the method comprises mutagenizing the first nucleic acidof step (a) by a method comprising an optimized directed evolutionsystem, gene site-saturation mutagenesis (GSSM™), synthetic ligationreassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly(GeneReassembly™, U.S. Pat. No. 6,537,776), gene site saturatedmutagenesis (GSSM™), synthetic ligation reassembly (SLR) and acombination thereof. In another aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises a xylanase enzyme encoded by a nucleic acid of theinvention; (b) providing a substrate for at least one of the enzymes ofstep (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions. Theinvention provides methods for modifying a small molecule comprising thefollowing steps: (a) providing a xylanase enzyme, wherein the enzymecomprises a polypeptide of the invention, or, a polypeptide encoded by anucleic acid of the invention, or a subsequence thereof; (b) providing asmall molecule; and (c) reacting the enzyme of step (a) with the smallmolecule of step (b) under conditions that facilitate an enzymaticreaction catalyzed by the xylanase enzyme, thereby modifying a smallmolecule by a xylanase enzymatic reaction. In one aspect, the method cancomprise a plurality of small molecule substrates for the enzyme of step(a), thereby generating a library of modified small molecules producedby at least one enzymatic reaction catalyzed by the xylanase enzyme. Inone aspect, the method can comprise a plurality of additional enzymesunder conditions that facilitate a plurality of biocatalytic reactionsby the enzymes to form a library of modified small molecules produced bythe plurality of enzymatic reactions. In another aspect, the method canfurther comprise the step of testing the library to determine if aparticular modified small molecule that exhibits a desired activity ispresent within the library. The step of testing the library can furthercomprise the steps of systematically eliminating all but one of thebiocatalytic reactions used to produce a portion of the plurality of themodified small molecules within the library by testing the portion ofthe modified small molecule for the presence or absence of theparticular modified small molecule with a desired activity, andidentifying at least one specific biocatalytic reaction that producesthe particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment ofa xylanase enzyme comprising the steps of: (a) providing a xylanaseenzyme, wherein the enzyme comprises a polypeptide of the invention, ora polypeptide encoded by a nucleic acid of the invention, or asubsequence thereof, and (b) deleting a plurality of amino acid residuesfrom the sequence of step (a) and testing the remaining subsequence fora xylanase activity, thereby determining a functional fragment of axylanase enzyme. In one aspect, the xylanase activity is measured byproviding a xylanase substrate and detecting a decrease in the amount ofthe substrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods of increasing thermotolerance orthermostability of a xylanase polypeptide, the method comprisingglycosylating a xylanase polypeptide, wherein the polypeptide comprisesat least thirty contiguous amino acids of a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid sequence of theinvention, thereby increasing the thermotolerance or thermostability ofthe xylanase polypeptide. In one aspect, the xylanase specific activitycan be thermostable or thermotolerant at a temperature in the range fromgreater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinant xylanasepolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid of the invention or a nucleicacid sequence of the invention, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell. The invention provides methods of expressing aheterologous nucleic acid sequence in a plant cell comprising thefollowing steps: (a) transforming the plant cell with a heterologousnucleic acid sequence operably linked to a promoter, wherein theheterologous nucleic sequence comprises a sequence of the invention; (b)growing the plant under conditions wherein the heterologous nucleicacids sequence is expressed in the plant cell.

The invention provides methods for hydrolyzing, breaking up ordisrupting a xylan-comprising composition comprising the followingsteps: (a) providing a polypeptide of the invention having a xylanaseactivity, or a polypeptide encoded by a nucleic acid of the invention;(b) providing a composition comprising a xylan; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the xylanase hydrolyzes, breaks up or disrupts thexylan-comprising composition. In one aspect, the composition comprises aplant cell, a bacterial cell, a yeast cell, an insect cell, or an animalcell. Thus, the composition can comprise any plant or plant part, anyxylan-containing food or feed, a waste product and the like. Theinvention provides methods for liquefying or removing a xylan-comprisingcomposition comprising the following steps: (a) providing a polypeptideof the invention having a xylanase activity, or a polypeptide encoded bya nucleic acid of the invention; (b) providing a composition comprisinga xylan; and (c) contacting the polypeptide of step (a) with thecomposition of step (b) under conditions wherein the xylanase removes,softens or liquefies the xylan-comprising composition.

The invention provides detergent compositions comprising a polypeptideof the invention, or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide has a xylanase activity. The xylanasecan be a nonsurface-active xylanase or a surface-active xylanase. Thexylanase can be formulated in a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a gelform, a paste or a slurry form. The invention provides methods forwashing an object comprising the following steps: (a) providing acomposition comprising a polypeptide of the invention having a xylanaseactivity, or a polypeptide encoded by a nucleic acid of the invention;(b) providing an object; and (c) contacting the polypeptide of step (a)and the object of step (b) under conditions wherein the composition canwash the object.

The invention provides textiles or fabrics, including, e.g., threads,comprising a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention. In one aspect, the textiles or fabricscomprise xylan-containing fibers. The invention provides methods fortreating a textile or fabric (e.g., removing a stain from a composition)comprising the following steps: (a) providing a composition comprising apolypeptide of the invention having a xylanase activity, or apolypeptide encoded by a nucleic acid of the invention; (b) providing atextile or fabric comprising a xylan; and (c) contacting the polypeptideof step (a) and the composition of step (b) under conditions wherein thexylanase can treat the textile or fabric (e.g., remove the stain). Theinvention provides methods for improving the finish of a fabriccomprising the following steps: (a) providing a composition comprising apolypeptide of the invention having a xylanase activity, or apolypeptide encoded by a nucleic acid of the invention; (b) providing afabric; and (c) contacting the polypeptide of step (a) and the fabric ofstep (b) under conditions wherein the polypeptide can treat the fabricthereby improving the finish of the fabric. In one aspect, the fabric isa wool or a silk.

The invention provides feeds or foods comprising a polypeptide of theinvention, or a polypeptide encoded by a nucleic acid of the invention.The invention provides methods for hydrolyzing xylans in a feed or afood prior to consumption by an animal comprising the following steps:(a) obtaining a feed material comprising a xylanase of the invention, ora xylanase encoded by a nucleic acid of the invention; and (b) addingthe polypeptide of step (a) to the feed or food material in an amountsufficient for a sufficient time period to cause hydrolysis of the xylanand formation of a treated food or feed, thereby hydrolyzing the xylansin the food or the feed prior to consumption by the animal. In oneaspect, the invention provides methods for hydrolyzing xylans in a feedor a food after consumption by an animal comprising the following steps:(a) obtaining a feed material comprising a xylanase of the invention, ora xylanase encoded by a nucleic acid of the invention; (b) adding thepolypeptide of step (a) to the feed or food material; and (c)administering the feed or food material to the animal, wherein afterconsumption, the xylanase causes hydrolysis of xylans in the feed orfood in the digestive tract of the animal. The food or the feed can be,e.g., a cereal, a grain, a corn and the like.

The invention provides food or nutritional supplements for an animalcomprising a polypeptide of the invention, e.g., a polypeptide encodedby the nucleic acid of the invention. In one aspect, the polypeptide inthe food or nutritional supplement can be glycosylated. The inventionprovides edible enzyme delivery matrices comprising a polypeptide of theinvention, e.g., a polypeptide encoded by the nucleic acid of theinvention. In one aspect, the delivery matrix comprises a pellet. In oneaspect, the polypeptide can be glycosylated. In one aspect, the xylanaseactivity is thermotolerant. In another aspect, the xylanase activity isthermostable.

The invention provides a food, a feed or a nutritional supplementcomprising a polypeptide of the invention. The invention providesmethods for utilizing a xylanase as a nutritional supplement in ananimal diet, the method comprising: preparing a nutritional supplementcontaining a xylanase enzyme comprising at least thirty contiguous aminoacids of a polypeptide of the invention; and administering thenutritional supplement to an animal to increase utilization of a xylancontained in a feed or a food ingested by the animal. The animal can bea human, a ruminant or a monogastric animal. The xylanase enzyme can beprepared by expression of a polynucleotide encoding the xylanase in anorganism selected from the group consisting of a bacterium, a yeast, aplant, an insect, a fungus and an animal. The organism can be selectedfrom the group consisting of an S. pombe, S. cerevisiae, Pichiapastoris, Pseudomonas sp., E. coli, Streptomyces sp., Bacillus sp. andLactobacillus sp.

The invention provides edible enzyme delivery matrix comprising athermostable recombinant xylanase enzyme, e.g., a polypeptide of theinvention. The invention provides methods for delivering a xylanasesupplement to an animal, the method comprising: preparing an edibleenzyme delivery matrix in the form of pellets comprising a granulateedible carrier and a thermostable recombinant xylanase enzyme, whereinthe pellets readily disperse the xylanase enzyme contained therein intoaqueous media, and administering the edible enzyme delivery matrix tothe animal. The recombinant xylanase enzyme can comprise a polypeptideof the invention. The granulate edible carrier can comprise a carrierselected from the group consisting of a grain germ, a grain germ that isspent of oil, a hay, an alfalfa, a timothy, a soy hull, a sunflower seedmeal and a wheat midd. The edible carrier can comprise grain germ thatis spent of oil. The xylanase enzyme can be glycosylated to providethermostability at pelletizing conditions. The delivery matrix can beformed by pelletizing a mixture comprising a grain germ and a xylanase.The pelletizing conditions can include application of steam. Thepelletizing conditions can comprise application of a temperature inexcess of about 80° C. for about 5 minutes and the enzyme retains aspecific activity of at least 350 to about 900 units per milligram ofenzyme.

The invention provides methods for improving texture and flavor of adairy product comprising the following steps: (a) providing apolypeptide of the invention having a xylanase activity, or a xylanaseencoded by a nucleic acid of the invention; (b) providing a dairyproduct; and (c) contacting the polypeptide of step (a) and the dairyproduct of step (b) under conditions wherein the xylanase can improvethe texture or flavor of the dairy product. In one aspect, the dairyproduct comprises a cheese or a yogurt. The invention provides dairyproducts comprising a xylanase of the invention, or is encoded by anucleic acid of the invention.

The invention provides methods for improving the extraction of oil froman oil-rich plant material comprising the following steps: (a) providinga polypeptide of the invention having a xylanase activity, or a xylanaseencoded by a nucleic acid of the invention; (b) providing an oil-richplant material; and (c) contacting the polypeptide of step (a) and theoil-rich plant material. In one aspect, the oil-rich plant materialcomprises an oil-rich seed. The oil can be a soybean oil, an olive oil,a rapeseed (canola) oil or a sunflower oil.

The invention provides methods for preparing a fruit or vegetable juice,syrup, puree or extract comprising the following steps: (a) providing apolypeptide of the invention having a xylanase activity, or a xylanaseencoded by a nucleic acid of the invention; (b) providing a compositionor a liquid comprising a fruit or vegetable material; and (c) contactingthe polypeptide of step (a) and the composition, thereby preparing thefruit or vegetable juice, syrup, puree or extract.

The invention provides papers or paper products or paper pulp comprisinga xylanase of the invention, or a polypeptide encoded by a nucleic acidof the invention. The invention provides methods for treating a paper ora paper or wood pulp comprising the following steps: (a) providing apolypeptide of the invention having a xylanase activity, or a xylanaseencoded by a nucleic acid of the invention; (b) providing a compositioncomprising a paper or a paper or wood pulp; and (c) contacting thepolypeptide of step (a) and the composition of step (b) under conditionswherein the xylanase can treat the paper or paper or wood pulp. In oneaspect, the pharmaceutical composition acts as a digestive aid or ananti-microbial (e.g., against Salmonella). In one aspect, the treatmentis prophylactic. In one aspect, the invention provides oral careproducts comprising a polypeptide of the invention having a xylanaseactivity, or a xylanase encoded by a nucleic acid of the invention. Theoral care product can comprise a toothpaste, a dental cream, a gel or atooth powder, an odontic, a mouth wash, a pre- or post brushing rinseformulation, a chewing gum, a lozenge or a candy.

The invention provides contact lens cleaning compositions comprising apolypeptide of the invention having a xylanase activity, or a xylanaseencoded by a nucleic acid of the invention.

In one aspect, the invention provides methods for eliminating orprotecting animals from a microorganism comprising a xylan comprisingadministering a polypeptide of the invention. The microorganism can be abacterium comprising a xylan, e.g., Salmonella.

The invention provides an isolated nucleic acid having a sequence as setforth in SEQ ID NO.:189 and variants thereof having at least 50%sequence identity to SEQ ID NO.:189 and encoding polypeptides havingxylanase activity. In one aspect, the polypeptide has a xylanaseactivity, e.g., a thermostable xylanase activity.

The invention provides isolated or recombinant nucleic acids comprisingSEQ ID NO:189, wherein SEQ ID NO:189 comprises one or more or all of thefollowing sequence variations: the nucleotides at positions 22 to 24 areTTC, the nucleotides at positions 22 to 24 are TTT, the nucleotides atpositions 31 to 33 are CAC, the nucleotides at positions 31 to 33 areCAT, the nucleotides at positions 34 to 36 are TTG, the nucleotides atpositions 34 to 36 are TTA, the nucleotides at positions 34 to 36 areCTC, the nucleotides at positions 34 to 36 are CTT, the nucleotides atpositions 34 to 36 are CTA, the nucleotides at positions 34 to 36 areCTG, the nucleotides at positions 49 to 51 are ATA, the nucleotides atpositions 49 to 51 are ATT, the nucleotides at positions 49 to 51 areATC, the nucleotides at positions 178 to 180 are CAC, the nucleotides atpositions 178 to 180 are CAT, the nucleotides at positions 190 to 192are TGT, the nucleotides at positions 190 to 192 are TGC, thenucleotides at positions 190 to 192 are GTA, the nucleotides atpositions 190 to 192 are GTT, the nucleotides at positions 190 to 192are GTC, the nucleotides at positions 190 to 192 are GTG, thenucleotides at positions 193 to 195 are GTG, the nucleotides atpositions 193 to 195 are GTC, the nucleotides at positions 193 to 195are GTA, the nucleotides at positions 193 to 195 are GTT, thenucleotides at positions 202 to 204 are ATA, the nucleotides atpositions 202 to 204 are ATT, the nucleotides at positions 202 to 204are ATC, the nucleotides at positions 202 to 204 are GCT, thenucleotides at positions 202 to 204 are GCG, the nucleotides atpositions 202 to 204 are GCC, the nucleotides at positions 202 to 204are GCA, the nucleotides at positions 235 to 237 are CCA, thenucleotides at positions 235 to 237 are CCC, or the nucleotides atpositions 235 to 237 are CCG.

The invention provides isolated or recombinant polypeptides comprisingan amino acid sequence comprising SEQ ID NO:190, wherein SEQ ID NO:190comprises one or more or all of the following sequence variations: theaspartic acid at amino acid position 8 is phenylalanine, the glutamineat amino acid position 11 is histidine, the asparagine at amino acidposition 12 is leucine, the glycine at amino acid position 17 isisoleucine, the threonine at amino acid position 23 is threonine encodedby a codon other than the wild type codon, the glycine at amino acidposition 60 is histidine, the proline at amino acid position 64 iscysteine, the proline at amino acid position 64 is valine, the serine atamino acid position 65 is valine, the glycine at amino acid position 68is isoleucine, the glycine at amino acid position 68 is alanine, or theserine at amino acid position 79 is proline. In one aspect, thepolypeptide has a xylanase activity, e.g., a thermostable xylanaseactivity.

The invention provides isolated or recombinant nucleic acids comprisingSEQ ID NO:189, wherein SEQ ID NO:189 comprises one or more or allsequence variations set forth in Table 1 or Table 2. The inventionprovides isolated or recombinant polypeptides encoded by nucleic acidscomprising SEQ ID NO:189, wherein SEQ ID NO:189 comprises one or more orall sequence variations set forth in Table 1 or Table 2. In one aspect,the polypeptide has a xylanase activity, e.g., a thermostable xylanaseactivity.

The invention provides isolated or recombinant nucleic acids comprisingSEQ ID NO:379, wherein SEQ ID NO:379 comprises one or more or all of thefollowing sequence variations: the nucleotides at positions 22 to 24 areTTC, the nucleotides at positions 31 to 33 are CAC, the nucleotides atpositions 49 to 51 are ATA, the nucleotides at positions 178 to 180 areCAC, the nucleotides at positions 193 to 195 are GTG, the nucleotides atpositions 202 to 204 are GCT.

The invention provides isolated or recombinant polypeptides comprisingSEQ ID NO:380, wherein SEQ ID NO:380 comprises one or more or all of thefollowing sequence variations: D8F, Q11H, G17I, G60H, S65V and/or G68A.In one aspect, the polypeptide has a xylanase activity, e.g., athermostable xylanase activity.

The isolated or recombinant nucleic acids of the invention are alsoreferred to as “Group A nucleic acid sequences”. The invention providesan isolated nucleic acid including at least 10 consecutive bases of asequence as set forth in Group A nucleic acid sequences, sequencessubstantially identical thereto and the sequences complementary thereto.

The isolated or recombinant polypeptides of the invention, which includefunctional fragments of the exemplary sequences of the invention, arealso referred to as “Group B amino acid sequences”. Another aspect ofthe invention is an isolated or recombinant nucleic acid encoding apolypeptide having at least 10 consecutive amino acids of a sequence asset forth in Group B amino acid sequences and sequences substantiallyidentical thereto. In yet another aspect, the invention provides apurified polypeptide having a sequence as set forth in Group B aminoacid sequences and sequences substantially identical thereto. Anotheraspect of the invention is an isolated or purified antibody thatspecifically binds to a polypeptide having a sequence as set forth inGroup B amino acid sequences and sequences substantially identicalthereto.

Another aspect of the invention is an isolated or purified antibody orbinding fragment thereof, which specifically binds to a polypeptidehaving at least 10 consecutive amino acids of one of the polypeptides ofGroup B amino acid sequences and sequences substantially identicalthereto.

Another aspect of the invention is a method of making a polypeptidehaving a sequence as set forth in Group B amino acid sequences andsequences substantially identical thereto. The method includesintroducing a nucleic acid encoding the polypeptide into a host cell,wherein the nucleic acid is operably linked to a promoter and culturingthe host cell under conditions that allow expression of the nucleicacid. Another aspect of the invention is a method of making apolypeptide having at least 10 amino acids of a sequence as set forth inGroup B amino acid sequences and sequences substantially identicalthereto. The method includes introducing a nucleic acid encoding thepolypeptide into a host cell, wherein the nucleic acid is operablylinked to a promoter and culturing the host cell under conditions thatallow expression of the nucleic acid, thereby producing the polypeptide.

Another aspect of the invention is a method of generating a variantincluding obtaining a nucleic acid having a sequence as set forth inGroup A nucleic acid sequences, sequences substantially identicalthereto, sequences complementary to the sequences of Group A nucleicacid sequences, fragments comprising at least 30 consecutive nucleotidesof the foregoing sequences and changing one or more nucleotides in thesequence to another nucleotide, deleting one or more nucleotides in thesequence, or adding one or more nucleotides to the sequence.

Another aspect of the invention is a computer readable medium havingstored thereon a sequence as set forth in Group A nucleic acid sequencesand sequences substantially identical thereto, or a polypeptide sequenceas set forth in Group B amino acid sequences and sequences substantiallyidentical thereto.

Another aspect of the invention is a computer system including aprocessor and a data storage device wherein the data storage device hasstored thereon a sequence as set forth in Group A nucleic acid sequencesand sequences substantially identical thereto, or a polypeptide having asequence as set forth in Group B amino acid sequences and sequencessubstantially identical thereto.

Another aspect of the invention is a method for comparing a firstsequence to a reference sequence wherein the first sequence is a nucleicacid having a sequence as set forth in Group A nucleic acid sequencesand sequences substantially identical thereto, or a polypeptide code ofGroup B amino acid sequences and sequences substantially identicalthereto. The method includes reading the first sequence and thereference sequence through use of a computer program that comparessequences; and determining differences between the first sequence andthe reference sequence with the computer program.

Another aspect of the invention is a method for identifying a feature ina sequence as set forth in Group A nucleic acid sequences and sequencessubstantially identical thereto, or a polypeptide having a sequence asset forth in Group B amino acid sequences and sequences substantiallyidentical thereto, including reading the sequence through the use of acomputer program which identifies features in sequences; and identifyingfeatures in the sequence with the computer program.

Yet another aspect of the invention is a method of catalyzing thebreakdown of xylan or a derivative thereof, comprising the step ofcontacting a sample containing xylan or the derivative thereof with apolypeptide of Group B amino acid sequences and sequences substantiallyidentical thereto under conditions which facilitate the breakdown of thexylan.

Another aspect of the invention is an assay for identifying fragments orvariants of Group B amino acid sequences and sequences substantiallyidentical thereto, which retain the enzymatic function of thepolypeptides of Group B amino acid sequences and sequences substantiallyidentical thereto. The assay includes contacting the polypeptide ofGroup B amino acid sequences, sequences substantially identical thereto,or polypeptide fragment or variant with a substrate molecule underconditions which allow the polypeptide fragment or variant to functionand detecting either a decrease in the level of substrate or an increasein the level of the specific reaction product of the reaction betweenthe polypeptide and substrate thereby identifying a fragment or variantof such sequences.

Another aspect of the invention is a nucleic acid probe of anoligonucleotide from about 10 to 50 nucleotides in length and having asegment of at least 10 contiguous nucleotides that is at least 50%complementary to a nucleic acid target region of a nucleic acid sequenceselected from the group consisting of Group A nucleic acid sequences;and which hybridizes to the nucleic acid target region under moderate tohighly stringent conditions to form a detectable target:probe duplex.

Another aspect of the invention is a polynucleotide probe for isolationor identification of xylanase genes having a sequence which is the sameas, or fully complementary to at least a fragment of one of Group Anucleic acid sequences.

In still another aspect, the invention provides a protein preparationcomprising a polypeptide having an amino acid sequence selected fromGroup B amino acid sequences and sequences substantially identicalthereto wherein the protein preparation is a liquid.

Still another aspect of the invention provides a protein preparationcomprising a polypeptide having an amino acid sequence selected fromGroup B amino acid sequences and sequences substantially identicalthereto wherein the polypeptide is a solid.

Yet another aspect of the invention provides a method for modifyingsmall molecules, comprising the step of mixing at least one polypeptideencoded by a polynucleotide selected from Group A nucleic acidsequences, sequences substantially identical thereto and the sequencescomplementary thereto with at least one small molecule, to produce atleast one modified small molecule via at least one biocatalyticreaction, where the at least one polypeptide has xylanase activity.

Another aspect of the invention is a cloning vector of a sequence thatencodes a polypeptide having xylanase activity, said sequence beingselected from Group A nucleic acid sequences, sequences substantiallyidentical thereto and the sequences complementary thereto.

Another aspect of the invention is a host cell comprising a sequencethat encodes a polypeptide having xylanase activity, said sequence beingselected from Group A nucleic acid sequences, sequences substantiallyidentical thereto and the sequences complementary thereto.

In yet another aspect, the invention provides an expression vectorcapable of replicating in a host cell comprising a polynucleotide havinga sequence selected Group A nucleic acid sequences, sequencessubstantially identical thereto, sequences complementary thereto andisolated nucleic acids that hybridize to nucleic acids having any of theforegoing sequences under conditions of low, moderate and highstringency.

In another aspect, the invention provides a method of dough conditioningcomprising contacting dough with at least one polypeptide of Group Bamino acid sequences and sequences substantially identical thereto underconditions sufficient for conditioning the dough.

Another aspect of the invention is a method of beverage productioncomprising administration of at least one polypeptide of Group B aminoacid sequences and sequences substantially identical thereto underconditions sufficient for decreasing the viscosity of wort or beer.

The xylanases of the invention are used to break down the high molecularweight arabinoxylans in animal feed. Adding the xylanases of theinvention stimulates growth rates by improving digestibility, which alsoimproves the quality of the animal litter. Xylanase functions throughthe gastro-intestinal tract to reduce intestinal viscosity and increasediffusion of pancreatic enzymes. Additionally, the xylanases of theinvention may be used in the treatment of endosperm cell walls of feedgrains and vegetable proteins. In one aspect of the invention, the novelxylanases of the invention are administered to an animal in order toincrease the utilization of the xylan in the food. This activity of thexylanases of the invention may be used to break down insoluble cell wallmaterial, liberating nutrients in the cell walls, which then becomeavailable to the animal. It also changes hemicellulose to nutritivesugars so that nutrients formerly trapped within the cell walls arereleased. Xylanase also produces compounds that may be a nutritivesource for the ruminal microflora.

Another aspect of the invention provides a method for utilizing xylanaseas a nutritional supplement in the diets of animals, comprisingpreparation of a nutritional supplement containing a recombinantxylanase enzyme comprising at least thirty contiguous amino acids ofGroup B amino acid sequences and sequences substantially identicalthereto and administering the nutritional supplement to an animal toincrease the utilization of xylan contained in food ingested by theanimal.

In another aspect of the invention, a method for delivering a xylanasesupplement to an animal is provided, where the method comprisespreparing an edible enzyme delivery matrix in the form of pelletscomprising a granulate edible carrier and a thermostable recombinantxylanase enzyme, wherein the particles readily disperse the xylanaseenzyme contained therein into aqueous media, and administering theedible enzyme delivery matrix to the animal. The granulate ediblecarrier may comprise a carrier selected from the group consisting ofgrain germ that is spent of oil, hay, alfalfa, timothy, soy hull,sunflower seed meal and wheat midd. The xylanase enzyme may have anamino acid sequence as set forth in Group B amino acid sequences andsequences substantially identical thereto.

In another aspect, the invention provides an isolated nucleic acidcomprising a sequence that encodes a polypeptide having xylanaseactivity, wherein the sequence is selected from Group A nucleic acidsequences, sequences substantially identical thereto and the sequencescomplementary thereto, wherein the sequence contains a signal sequence.The invention also provides an isolated nucleic acid comprising asequence that encodes a polypeptide having xylanase activity, whereinthe sequence is selected from Group A nucleic acid sequences, sequencessubstantially identical thereto and the sequences complementary thereto,wherein the sequence contains a signal sequence from another xylanase.Additionally, the invention provides an isolated nucleic acid comprisinga sequence that encodes a polypeptide having xylanase activity, whereinthe sequence is selected from Group A nucleic acid sequences, sequencessubstantially identical thereto and the sequences complementary theretowherein the sequence does not contain a signal sequence.

Still another aspect of the invention provides an isolated nucleic acidthat is a mutation of SEQ ID NO:189. Yet another aspect provides anamino acid sequence that is a mutation of SEQ ID NO:190.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of aspects of the invention andare not meant to limit the scope of the invention as encompassed by theclaims.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 is a graph comparing activity of the wild type sequence (SEQ IDNOS: 189 and 190) to the 8x mutant (SEQ ID NOS:375, 376), a combinationof mutants D, F, H, I, S, V, X and AA in Table 1.

FIG. 6A illustrates the nine single site amino acid mutants of SEQ IDNO:378 (encoded by SEQ ID NO:377) as generated by Gene Site SaturationMutagenesis (GSSM™) of SEQ ID NO:190 (encoded by SEQ ID NO:189), asdescribed in detail in Example 5, below.

FIG. 6B illustrates the unfolding of SEQ ID NO:190 and SEQ ID NO:378 inmelting temperature transition midpoint (Tm) experiments as determinedby DSC for each enzyme, as described in detail in Example 5, below.

FIG. 7A illustrates the pH and temperature activity profiles for theenzymes SEQ ID NO:190 and SEQ ID NO:378, as described in detail inExample 5, below.

FIG. 7B illustrates the rate/temperature activity optima for the enzymesSEQ ID NO:190 and SEQ ID NO:378, as described in detail in Example 5,below.

FIG. 7C illustrates the thermal tolerance/residual activity for theenzymes SEQ ID NO:190 and SEQ ID NO:378, as described in detail inExample 5, below.

FIG. 8A illustrates the GeneReassembly™ library of all possiblecombinations of the 9 GSSM™ point mutations that was constructed andscreened for variants with improved thermal tolerance and activity, asdescribed in detail in Example 5, below.

FIG. 8B illustrates the relative activity of the “6X-2” variant and “9X”variant (SEQ ID NO:378) compared to SEQ ID NO:190 (“wild-type”) at atemperature optimum and pH 6.0, as described in detail in Example 5,below.

FIG. 9A illustrates the fingerprints obtained after hydrolysis ofoligoxylans (Xyl)3, (Xyl)4, (Xyl)5 and (Xyl)6 by the SEQ ID NO:190(“wild-type”) and the “9X” variant (SEQ ID NO:378) enzymes, as describedin detail in Example 5, below.

FIG. 9B illustrates the fingerprints obtained after hydrolysis ofBeechwood xylan by the SEQ ID NO:190 (“wild-type”) and the “9X” variant(SEQ ID NO:378) enzymes, as described in detail in Example 5, below.

FIG. 10A is a schematic diagram illustrating the level of thermalstability (represented by Tm) improvement over SEQ ID NO:190(“wild-type”) obtained by GSSM™ evolution, as described in detail inExample 5, below.

FIG. 10B illustrates a “fitness diagram” of enzyme improvement in theform of SEQ ID NO:378 and SEQ ID NO:380, as obtained by combining GSSM™and GeneReassembly™ technologies, as described in detail in Example 5,below.

FIG. 11 is a schematic flow diagram of an exemplary routine screeningprotocol to determine whether a xylanase of the invention is useful inpretreating paper pulp, as described in detail in Example 6, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to xylanases and polynucleotides encodingthem and methods of making and using them. Xylanase activity of thepolypeptides of the invention encompasses enzymes having hydrolaseactivity, for example, enzymes capable of hydrolyzing glycosidiclinkages present in xylan, e.g., catalyzing hydrolysis of internalβ-1,4-xylosidic linkages. The xylanases of the invention can be used tomake and/or process foods, feeds, nutritional supplements, textiles,detergents and the like. The xylanases of the invention can be used inpharmaceutical compositions and dietary aids. Xylanases of the inventionare particularly useful in baking, animal feed, beverage and paperprocesses.

Definitions

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin. Thephrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double strandediRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. “Oligonucleotide” includeseither a single stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands that may be chemically synthesized. Suchsynthetic oligonucleotides have no 5′ phosphate and thus will not ligateto another oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide can ligate to afragment that has not been dephosphorylated.

A “coding sequence of” or a “nucleotide sequence encoding” a particularpolypeptide or protein, is a nucleic acid sequence which is transcribedand translated into a polypeptide or protein when placed under thecontrol of appropriate regulatory sequences.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening sequences (introns) between individual coding segments(exons). “Operably linked” as used herein refers to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments.Typically, it refers to the functional relationship of transcriptionalregulatory sequence to a transcribed sequence. For example, a promoteris operably linked to a coding sequence, such as a nucleic acid of theinvention, if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as a xylanase of the invention)in a host compatible with such sequences. Expression cassettes includeat least a promoter operably linked with the polypeptide codingsequence; and, optionally, with other sequences, e.g., transcriptiontermination signals. Additional factors necessary or helpful ineffecting expression may also be used, e.g., enhancers. Thus, expressioncassettes also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like. A“vector” comprises a nucleic acid that can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors that ensure that genes encoding proteins specific to agiven tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. As used herein, the term“transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, e.g., the nucleicacids and various recombinant constructs (e.g., expression cassettes) ofthe invention.

“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these and to naturally occurringor synthetic molecules.

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterm “polypeptide” as used herein, refers to amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres and may contain modified amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Modificationscan occur anywhere in the polypeptide, including the peptide backbone,the amino acid side-chains and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also agiven polypeptide may have many types of modifications. Modificationsinclude acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment of aphosphytidylinositol, cross-linking cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristolyation, oxidation, pegylation, xylanhydrolase processing, phosphorylation, prenylation, racemization,selenoylation, sulfation and transfer-RNA mediated addition of aminoacids to protein such as arginylation. (See Creighton, T. E.,Proteins—Structure and Molecular Properties 2nd Ed., W.H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12(1983)). The peptides and polypeptides of the invention also include all“mimetic” and “peptidomimetic” forms, as described in further detail,below.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 10⁴-10⁶ fold. However, the term “purified” also includes nucleicacids that have been purified from the remainder of the genomic DNA orfrom other sequences in a library or other environment by at least oneorder of magnitude, typically two or three orders and more typicallyfour or five orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid isadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. Additionally, to be “enriched” the nucleic acidswill represent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid backbone molecules. Backbone moleculesaccording to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. Typically, the enriched nucleic acidsrepresent 15% or more of the number of nucleic acid inserts in thepopulation of recombinant backbone molecules. More typically, theenriched nucleic acids represent 50% or more of the number of nucleicacid inserts in the population of recombinant backbone molecules. In aone aspect, the enriched nucleic acids represent 90% or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules.

“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; i.e., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis. Solid-phase chemical peptide synthesismethods can also be used to synthesize the polypeptide or fragments ofthe invention. Such method have been known in the art since the early1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (Seealso Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recentlybeen employed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate. When such a systemis utilized, a plate of rods or pins is inverted and inserted into asecond plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A automated peptidesynthesizer. Such equipment provides ready access to the peptides of theinvention, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

A promoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter will transcribethe coding sequence into mRNA.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion, gel electrophoresis may beperformed to isolate the desired fragment.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, refers to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one of the known sequencecomparison algorithms or by visual inspection. Typically, thesubstantial identity exists over a region of at least about 100 residuesand most commonly the sequences are substantially identical over atleast about 150-200 residues. In some aspects, the sequences aresubstantially identical over the entire length of the coding regions.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule and provided that the polypeptideessentially retains its functional properties. A conservative amino acidsubstitution, for example, substitutes one amino acid for another of thesame class (e.g., substitution of one hydrophobic amino acid, such asisoleucine, valine, leucine, or methionine, for another, or substitutionof one polar amino acid for another, such as substitution of argininefor lysine, glutamic acid for aspartic acid or glutamine forasparagine). One or more amino acids can be deleted, for example, from axylanase polypeptide, resulting in modification of the structure of thepolypeptide, without significantly altering its biological activity. Forexample, amino- or carboxyl-terminal amino acids that are not requiredfor xylanase biological activity can be removed. Modified polypeptidesequences of the invention can be assayed for xylanase biologicalactivity by any number of methods, including contacting the modifiedpolypeptide sequence with a xylanase substrate and determining whetherthe modified polypeptide decreases the amount of specific substrate inthe assay or increases the bioproducts of the enzymatic reaction of afunctional xylanase polypeptide with the substrate.

“Fragments” as used herein are a portion of a naturally occurringprotein which can exist in at least two different conformations.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein. “Substantially the same”means that an amino acid sequence is largely, but not entirely, thesame, but retains at least one functional activity of the sequence towhich it is related. In general two amino acid sequences are“substantially the same” or “substantially homologous” if they are atleast about 85% identical. Fragments which have different threedimensional structures as the naturally occurring protein are alsoincluded. An example of this, is a “pro-form” molecule, such as a lowactivity proprotein that can be modified by cleavage to produce a matureenzyme with significantly higher activity.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature and arewell known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature. In alternativeaspects, nucleic acids of the invention are defined by their ability tohybridize under various stringency conditions (e.g., high, medium, andlow), as set forth herein.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDSand 200 n/ml sheared and denatured salmon sperm DNA. Hybridization couldoccur under reduced stringency conditions as described above, but in 35%formamide at a reduced temperature of 35° C. The temperature rangecorresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “variant” refers to polynucleotides or polypeptides of theinvention modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of a xylanase of the invention. Variants can be produced by anynumber of means included methods such as, for example, error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (e.g., GeneReassembly™, see, e.g., U.S.Pat. No. 6,537,776), GSSM™ and any combination thereof.

Table 1 and Table 2 list variants obtained by mutating SEQ ID NO:189(encoding SEQ ID NO:190) by GSSM™. The invention provides nucleic acidshaving one or more, or all, of the sequences as set forth in Tables 1and 2, i.e., nucleic acids having sequences that are variants of SEQ IDNO:189, where the variations are set forth in Table 1 and Table 2, andthe polypeptides that are encoded by these variants.

These GSSM™ variants (set forth in Tables 1 and 2) were tested forthermal tolerance (see Examples, below). Mutants D, F, G, H, I, J, K, S,T, U, V, W, X, Y, Z, AA, DD and EE were found to have the highestthermal tolerance among the mutants in Table 1. Mutants may also becombined to form a larger mutant. For example, mutants D, F, H, I, S, V,X and AA of Table 1 were combined to form a larger mutant termed “8x”with a sequence as set forth in SEQ ID NO:375 (polypeptide encodingnucleic acid) and SEQ ID NO:376 (amino acid sequence). FIG. 5 is a graphcomparing the activity of the wild type sequence (SEQ ID NOS: 189 and190) to the 8x mutant (SEQ ID NOS: 259 and 260). In comparing the wildtype and the 8x mutant, it was discovered that the optimal temperaturefor both was 65° C. and that the optimal pH for both was 5.5. The wildtype sequence was found to maintain its stability for less than 1 minuteat 65° C., while the 8x mutant (SEQ ID NOS:375, 376) was found tomaintain its stability for more than 10 minutes at 85° C. The substrateused was AZO-AZO-xylan. In one aspect, the 8x mutant (SEQ ID NOS:375,376) was evolved by GSSM™. In another aspect, the wild type is a GSSM™parent for thermal tolerance evolution.

TABLE 1 Mutant Mutation Wild type Seq GSSM ™ Seq A A2F GCC TTT B A2D GCCGAC C A5H GCT CAC D D8F GAC TTC E Q11L CAA CTC F Q11H CAA CAC G N12L AATTTG H N12L AAT TTG I G17I GGT ATA J Q11H, T23T CAA, ACC CAT, ACG K Q11HCAA CAT L S26P TCT CCG M S26P TCT CCA N S35F TCA TTT O No Change GTT GTAP A51P GCA CCG Q A51P GCA CCG R G60R GGA CGC S G60H GGA CAC T G60H GGACAC U P64C CCG TGT V P64V CCG GTA W P64V CCG GTT X S65V TCC GTG Y Q11HCAA CAT Z G68I GGA ATA AA G68A GGA GCT BB A71G GCT GGA CC No Change AATAAC DD S79P TCA CCA EE S79P TCA CCC FF T95S ACT TCT GG Y98P TAT CCG HHT114S ACT AGC II No Change AAC AAC JJ No Change AGG AGA KK I142L ATT CTGLL S151I AGC ATC MM S138T, S151A TCG, AGC ACG, GCG NN K158R AAG CGG OOK160V, V172I AAA, GTA GTT, ATA

The codon variants as set forth in Table 2 that produced variants (ofSEQ ID NO:189) with the best variation or “improvement” over “wild type”(SEQ ID NO:189) in thermal tolerance are highlighted. As noted above,the invention provides nucleic acids, and the polypeptides that encodethem, comprising one, several or all or the variations set forth inTable 2 and Table 1.

TABLE 2 Wild type GSSM ™ Other codons also coding for Mutation SequenceSequence same changed amino acid A2F GCC TTT TTC A2D GCC GAC GAT A5H GCTCAC CAT

 GAC

 TTT Q11L CAA CTC TTA, TTG, CTT, CTA, CTG

 CAA

 —

 AAT

 TTA, CTC, CTT, CTA, CTG

 GGT

 ATT, ATC T23T ACC ACG ACT, ACC, ACA S26P TCT CCG, CCA CCC S35F TCA TTTTTC A51P GCA CCG CCC, CCA G60R GGA CGC CGT, CGA, CGG, AGA, AGG

 GGA

 CAT

 CCG

 TGC

 CCG

 GTC, GTG

 TCC

 GTC, GTA, GTT

 GGA

 ATT, ATC

 GGA

 GCG, GCC, GCA A71G GCT GGA GGT, GGC, GGG

 TCA

 CCG T95S ACT TCT TCC, TCA, TCG, AGT, AGC Y98P TAT CCG CCC, CCA T114SACT AGC TCC, TCA, TCG, AGT, TCT I142L ATT CTG TTA, CTC, CTT, CTA, TTGS151I AGC ATC ATT, ATA S138T TCG ACG ACT, ACC, ACA S151A AGC GCG GCT,GCC, GCA K158R AAG CGG CGT, CGA, CGC, AGA, AGG K160V AAA GTT GTC, GTA,GTG V172I GTA ATA ATT, ATC

In one aspect the amino acid sequence of an amino acid sequence (SEQ IDNO: 208) of Group B amino acid sequences is modified by a single aminoacid mutation. In a specific aspect, that mutation is an asparagine toaspartic acid mutation. The resulting amino acid sequence andcorresponding nucleic acid sequence are set forth as SEQ ID NO:252 andSEQ ID NO:251, respectively. Single amino acid mutations with animprovement in the pH optimum of the enzyme, such as the mutation of SEQID NO:208, have been shown in the art with respect to xylanases. (See,for example, Joshi, M., Sidhu, G., Pot, I., Brayer, G., Withers, S.,McIntosh, L., J. Mol. Bio. 299, 255-279 (2000).) It is also noted thatin such single amino acid mutations, portions of the sequences may beremoved in the subcloning process. For example, SEQ ID NO:207 and SEQ IDNO:251 differ in only one nucleotide, over the area that the sequencesalign. However, it is noted that a 78 nucleotide area at the N-terminusof SEQ ID NO:207 was removed from the N-terminus of SEQ ID NO:251 in thesubcloning. Additionally, the first three nucleotides in SEQ ID NO:251were changed to ATG and then the point mutation was made at the sixthnucleotide in SEQ ID NO:251.

The term “saturation mutagenesis”, “gene site saturated mutagenesis” or“GSSM™” includes a method that uses degenerate oligonucleotide primersto introduce point mutations into a polynucleotide, as described indetail, below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids (e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ IDNO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,SEQ ID NO:377 or SEQ ID NO:379; nucleic acids encoding polypeptides asset forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ IDNO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166,SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ IDNO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194,SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ IDNO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222,SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ IDNO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250,SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ IDNO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278,SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ IDNO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306,SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ IDNO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334,SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ IDNO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362,SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ IDNO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378 or SEQ ID NO:380),including expression cassettes such as expression vectors, encoding thepolypeptides of the invention. The invention also includes methods fordiscovering new xylanase sequences using the nucleic acids of theinvention. The invention also includes methods for inhibiting theexpression of xylanase genes, transcripts and polypeptides using thenucleic acids of the invention. Also provided are methods for modifyingthe nucleic acids of the invention by, e.g., synthetic ligationreassembly, optimized directed evolution system and/or saturationmutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Forexample, the following exemplary sequences of the invention wereinitially derived from the following sources:

TABLE 3 SEQ ID SOURCE 1, 2 Bacteria 101, 102 Environmental 103, 104Bacteria 105, 106 Environmental 107, 108 Bacteria 109, 110 Environmental11, 12 Environmental 111, 112 Environmental 113, 114 Environmental 115,116 Environmental 117, 118 Environmental 119, 120 Environmental 121, 122Environmental 123, 124 Environmental 125, 126 Environmental 127, 128Environmental 129, 130 Bacteria 13, 14 Environmental 131, 132Environmental 133, 134 Environmental 135, 136 Environmental 137, 138Environmental 139, 140 Environmental 141, 142 Environmental 143, 144Bacteria 145, 146 Eukaryote 147, 148 Environmental 149, 150Environmental 15, 16 Environmental 151, 152 Environmental 153, 154Environmental 155, 156 Environmental 157, 158 Environmental 159, 160Environmental 161, 162 Environmental 163, 164 Environmental 165, 166Environmental 167, 168 Environmental 169, 170 Environmental 17, 18Bacteria 171, 172 Environmental 173, 174 Environmental 175, 176Environmental 177, 178 Environmental 179, 180 Environmental 181, 182Environmental 183, 184 Environmental 185, 186 Environmental 187, 188Environmental 189, 190 Environmental 19, 20 Environmental 191, 192Environmental 193, 194 Environmental 195, 196 Environmental 197, 198Environmental 199, 200 Environmental 201, 202 Environmental 203, 204Environmental 205, 206 Environmental 207, 208 Environmental 209, 210Environmental 21, 22 Environmental 211, 212 Environmental 213, 214Environmental 215, 216 Environmental 217, 218 Environmental 219, 220Environmental 221, 222 Environmental 223, 224 Environmental 225, 226Environmental 227, 228 Environmental 229, 230 Environmental 23, 24Environmental 231, 232 Bacteria 233, 234 Environmental 235, 236Environmental 237, 238 Environmental 239, 240 Environmental 241, 242Environmental 243, 244 Environmental 245, 246 Environmental 247, 248Environmental 249, 250 Environmental 25, 26 Environmental 251, 252Environmental 253, 254 Environmental 255, 256 Environmental 257, 258Environmental 259, 260 Environmental 261, 262 Environmental 263, 264Environmental 265, 266 Environmental 267, 268 Bacteria 269, 270Environmental 27, 28 Environmental 271, 272 Environmental 273, 274Environmental 275, 276 Environmental 277, 278 Environmental 279, 280Environmental 281, 282 Environmental 283, 284 Environmental 285, 286Environmental 287, 288 Environmental 289, 290 Environmental 29, 30Archaea 291, 292 Environmental 293, 294 Environmental 295, 296Environmental 297, 298 Environmental 299, 300 Environmental 3, 4Environmental 301, 302 Environmental 303, 304 Environmental 305, 306Bacteria 307, 308 Environmental 309, 310 Environmental 31, 32Environmental 311, 312 Environmental 313, 314 Bacteria 315, 316Environmental 317, 318 Environmental 319, 320 Environmental 321, 322Environmental 323, 324 Environmental 325, 326 Environmental 327, 328Environmental 329, 330 Environmental 33, 34 Environmental 331, 332Environmental 333, 334 Environmental 335, 336 Environmental 337, 338Environmental 339, 340 Environmental 341, 342 Environmental 343, 344Environmental 345, 346 Environmental 347, 348 Environmental 349, 350Environmental 35, 36 Environmental 351, 352 Environmental 353, 354Environmental 355, 356 Environmental 357, 358 Environmental 359, 360Environmental 361, 362 Environmental 363, 364 Environmental 365, 366Environmental 367, 368 Environmental 369, 370 Environmental 37, 38Environmental 371, 372 Environmental 373, 374 Environmental 375, 376Artificial 377, 378 Artificial 39, 40 Environmental 41, 42 Environmental43, 44 Environmental 45, 46 Environmental 47, 48 Environmental 49, 50Environmental 5, 6 Environmental 51, 52 Environmental 53, 54 Bacteria55, 56 Environmental 57, 58 Environmental 59, 60 Environmental 61, 62Environmental 63, 64 Environmental 65, 66 Environmental 67, 68Environmental 69, 70 Environmental 7, 8 Environmental 71, 72Environmental 73, 74 Environmental 75, 76 Environmental 77, 78Environmental 79, 80 Environmental 81, 82 Environmental 83, 84Environmental 85, 86 Bacteria 87, 88 Environmental 89, 90 Bacteria 9, 10Environmental 91, 92 Environmental 93, 94 Environmental 95, 96Environmental 97, 98 Environmental 99, 100 Environmental

In one aspect, the invention also provides xylanase-encoding nucleicacids with a common novelty in that they are derived from anenvironmental source, or a bacterial source, or an archaeal source.

In practicing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

One aspect of the invention is an isolated nucleic acid comprising oneof the sequences of Group A nucleic acid sequences and sequencessubstantially identical thereto, the sequences complementary thereto, ora fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 consecutive bases of one of the sequences ofa Group A nucleic acid sequence (or the sequences complementarythereto). The isolated, nucleic acids may comprise DNA, including cDNA,genomic DNA and synthetic DNA. The DNA may be double-stranded orsingle-stranded and if single stranded may be the coding strand ornon-coding (anti-sense) strand. Alternatively, the isolated nucleicacids may comprise RNA.

As discussed in more detail below, the isolated nucleic acids of one ofthe Group A nucleic acid sequences and sequences substantially identicalthereto, may be used to prepare one of the polypeptides of a Group Bamino acid sequence and sequences substantially identical thereto, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids of one of the polypeptides of GroupB amino acid sequences and sequences substantially identical thereto.

Accordingly, another aspect of the invention is an isolated nucleic acidwhich encodes one of the polypeptides of Group B amino acid sequencesand sequences substantially identical thereto, or fragments comprisingat least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids of one of the polypeptides of the Group B amino acidsequences. The coding sequences of these nucleic acids may be identicalto one of the coding sequences of one of the nucleic acids of Group Anucleic acid sequences, or a fragment thereof or may be different codingsequences which encode one of the polypeptides of Group B amino acidsequences, sequences substantially identical thereto and fragmentshaving at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids of one of the polypeptides of Group B amino acidsequences, as a result of the redundancy or degeneracy of the geneticcode. The genetic code is well known to those of skill in the art andcan be obtained, for example, on page 214 of B. Lewin, Genes VI, OxfordUniversity Press, 1997.

The isolated nucleic acid which encodes one of the polypeptides of GroupB amino acid sequences and sequences substantially identical thereto,may include, but is not limited to: only the coding sequence of one ofGroup A nucleic acid sequences and sequences substantially identicalthereto and additional coding sequences, such as leader sequences orproprotein sequences and non-coding sequences, such as introns ornon-coding sequences 5, and/or 3′ of the coding sequence. Thus, as usedherein, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only the coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

Alternatively, the nucleic acid sequences of Group A nucleic acidsequences and sequences substantially identical thereto, may bemutagenized using conventional techniques, such as site directedmutagenesis, or other techniques familiar to those skilled in the art,to introduce silent changes into the polynucleotides of Group A nucleicacid sequences and sequences substantially identical thereto. As usedherein, “silent changes” include, for example, changes which do notalter the amino acid sequence encoded by the polynucleotide. Suchchanges may be desirable in order to increase the level of thepolypeptide produced by host cells containing a vector encoding thepolypeptide by introducing codons or codon pairs which occur frequentlyin the host organism.

The invention also relates to polynucleotides which have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides of Group B amino acidsequences and sequences substantially identical thereto. Such nucleotidechanges may be introduced using techniques such as site directedmutagenesis, random chemical mutagenesis, exonuclease III deletion andother recombinant DNA techniques. Alternatively, such nucleotide changesmay be naturally occurring allelic variants which are isolated byidentifying nucleic acids which specifically hybridize to probescomprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200,300, 400, or 500 consecutive bases of one of the sequences of Group Anucleic acid sequences and sequences substantially identical thereto (orthe sequences complementary thereto) under conditions of high, moderate,or low stringency as provided herein.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., xylanases) generated from these nucleicacids can be individually isolated or cloned and tested for a desiredactivity. Any recombinant expression system can be used, includingbacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS 1N MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s). e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lac, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lacI promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused. Promoters suitable for expressing the polypeptide or fragmentthereof in bacteria include the E. coli lac or trp promoters, the lacIpromoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gptpromoter, the lambda P_(R) promoter, the lambda P_(L) promoter,promoters from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter.Fungal promoters include the a factor promoter. Eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, heat shock promoters, the early and late SV40 promoter, LTRsfrom retroviruses and the mouse metallothionein-I promoter. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express a xylanase of theinvention in a tissue-specific manner. The invention also providesplants or seeds that express a xylanase of the invention in atissue-specific manner. The tissue-specificity can be seed specific,stem specific, leaf specific, root specific, fruit specific and thelike.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPcl frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol. 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassaya vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression ofxylanase-expressing nucleic acid in a specific tissue, organ or celltype (i.e. tissue-specific promoters) or may be otherwise under moreprecise environmental or developmental control or under the control ofan inducible promoter. Examples of environmental conditions that mayaffect transcription include anaerobic conditions, elevated temperature,the presence of light, or sprayed with chemicals/hormones. For example,the invention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fb12A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant. MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the xylanase-producing nucleic acids of the invention willallow the grower to select plants with the optimal xylanase expressionand/or activity. The development of plant parts can thus controlled. Inthis way the invention provides the means to facilitate the harvestingof plants and plant parts. For example, in various embodiments, themaize In2-2 promoter, activated by benzenesulfonamide herbicidesafeners, is used (De Veylder (1997) Plant Cell Physiol. 38:568-577);application of different herbicide safeners induces distinct geneexpression patterns, including expression in the root, hydathodes, andthe shoot apical meristem. Coding sequences of the invention are alsounder the control of a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324).

In some aspects, proper polypeptide expression may requirepolyadenylation region at the 3′-end of the coding region. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant (or animal or other) genes, or from genes in theAgrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding thexylanases of the invention. Expression vectors and cloning vehicles ofthe invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen), pBluescriptplasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, anyother plasmid or other vector may be used so long as they are replicableand viable in the host. Low copy number or high copy number vectors maybe employed with the present invention.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant. Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences and 5′flanking nontranscribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin and theadenovirus enhancers.

In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli and the S. cerevisiae TRP1 gene.

In some aspects, the nucleic acid encoding one of the polypeptides ofGroup B amino acid sequences and sequences substantially identicalthereto, or fragments comprising at least about 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 consecutive amino acids thereof is assembledin appropriate phase with a leader sequence capable of directingsecretion of the translated polypeptide or fragment thereof. Optionally,the nucleic acid can encode a fusion polypeptide in which one of thepolypeptides of Group B amino acid sequences and sequences substantiallyidentical thereto, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof is fusedto heterologous peptides or polypeptides, such as N-terminalidentification peptides which impart desired characteristics, such asincreased stability or simplified purification.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculoviris, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, N.Y., (1989).

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a xylanase of theinvention, or a vector of the invention. The host cell may be any of thehost cells familiar to those skilled in the art, including prokaryoticcells, eukaryotic cells, such as bacterial cells, fungal cells, yeastcells, mammalian cells, insect cells, or plant cells. Exemplarybacterial cells include E. coli, Streptomyces, Bacillus subtilis,Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See, e.g., Weising (1988) Ann. Rev. Genet.22:421-477; U.S. Pat. No. 5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are can be used.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Host cells containing the polynucleotides of interest, e.g., nucleicacids of the invention, can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression and will be apparent to the ordinarilyskilled artisan. The clones which are identified as having the specifiedenzyme activity may then be sequenced to identify the polynucleotidesequence encoding an enzyme having the enhanced activity.

The invention provides a method for overexpressing a recombinantxylanase in a cell comprising expressing a vector comprising a nucleicacid of the invention, e.g., a nucleic acid comprising a nucleic acidsequence with at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a sequence of Group A nucleic acid sequencesover a region of at least about 100 residues, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by visual inspection, or, a nucleic acid that hybridizesunder stringent conditions to a nucleic acid sequence as set forth inGroup A nucleic acid sequences, or a subsequence thereof. Theoverexpression can be effected by any means, e.g., use of a highactivity promoter, a dicistronic vector or by gene amplification of thevector.

The nucleic acids of the invention can be expressed, or overexpressed,in any in vitro or in vivo expression system. Any cell culture systemscan be employed to express, or over-express, recombinant protein,including bacterial, insect, yeast, fungal or mammalian cultures.Over-expression can be effected by appropriate choice of promoters,enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors(see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun. 229:295-8),media, culture systems and the like. In one aspect, gene amplificationusing selection markers, e.g., glutamine synthetase (see, e.g., Sanders(1987) Dev. Biol. Stand. 66:55-63), in cell systems are used tooverexpress the polypeptides of the invention.

Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes EP 0659215(WO 9403612 A1) (Nevalainen et al.); Lapidot, A., Mechaly, A., Shoham,Y., “Overexpression and single-step purification of a thermostablexylanase from Bacillus stearothermophilus T-6,” J. Biotechnol. Nov51:259-64 (1996); Lüthi, E., Jasmat, N. B., Bergquist, P. L., “Xylanasefrom the extremely thermophilic bacterium Caldocellum saccharolyticum:overexpression of the gene in Escherichia coli and characterization ofthe gene product,” Appl. Environ. Microbiol. Sep 56:2677-83 (1990); andSung, W. L., Luk, C. K., Zahab, D. M., Wakarchuk, W., “Overexpression ofthe Bacillus subtilis and circulans xylanases in Escherichia coli,”Protein Expr. Purif. Jun 4:200-6 (1993), although these references donot teach the inventive enzymes of the instant application.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Salmonella typhimurium and variousspecies within the genera Pseudomonas, Streptomyces and Staphylococcus,fungal cells, such as yeast, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection. DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175, 1981) and other cell lines capable of expressing proteinsfrom a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK celllines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Alternatively, the polypeptides of Group B amino acid sequences andsequences substantially identical thereto, or fragments comprising atleast 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof can be synthetically produced by conventionalpeptide synthesizers. In other aspects, fragments or portions of thepolypeptides may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides of Group B amino acid sequences and sequences substantiallyidentical thereto, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof usingmRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the xylanases of the invention, or modified nucleic acidsof the invention, can be reproduced by amplification. Amplification canalso be used to clone or modify the nucleic acids of the invention.Thus, the invention provides amplification primer sequence pairs foramplifying nucleic acids of the invention. One of skill in the art candesign amplification primer sequence pairs for any part of or the fulllength of these sequences.

In one aspect, the invention provides a nucleic acid amplified by aprimer pair of the invention, e.g., a primer pair as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and about the first(the 5′) 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a xylanaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and a second memberhaving a sequence as set forth by about the first (the 5′) 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand of the first member. The invention providesxylanases generated by amplification, e.g., polymerase chain reaction(PCR), using an amplification primer pair of the invention. Theinvention provides methods of making a xylanase by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. In one aspect, the amplification primer pair amplifies anucleic acid from a library, e.g., a gene library, such as anenvironmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the invention(e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79,SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89,SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99,SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ IDNO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127,SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ IDNO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,SEQ ID NO:157, SEQ ID NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQ IDNO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183,SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ IDNO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211,SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ IDNO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239,SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ IDNO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267,SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ IDNO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295,SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ IDNO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323,SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ IDNO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351,SEQ ID NO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ IDNO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQID NO:371, SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377 or SEQ ID NO:379)over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,residues. The invention provides polypeptides comprising sequenceshaving at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to an exemplary polypeptide of theinvention. The extent of sequence identity (homology) may be determinedusing any computer program and associated parameters, including thosedescribed herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with thedefault parameters.

The nucleic acid sequences are also referred to as “Group A” nucleicacid sequences, which include sequences substantially identical thereto,as well as sequences homologous to Group A nucleic acid sequences andfragments thereof and sequences complementary to all of the precedingsequences. Nucleic acid sequences of the invention can comprise at least10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500consecutive nucleotides of an exemplary sequence of the invention (e.g.,Group A nucleic acid sequences) and sequences substantially identicalthereto. Homologous sequences and fragments of Group A nucleic acidsequences and sequences substantially identical thereto, refer to asequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, or 50% homology to these sequences. Homology may bedetermined using any of the computer programs and parameters describedherein, including FASTA version 3.0t78 with the default parameters.Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences as set forth in theGroup A nucleic acid sequences. The homologous sequences may be obtainedusing any of the procedures described herein or may result from thecorrection of a sequencing error. It will be appreciated that thenucleic acid sequences as set forth in Group A nucleic acid sequencesand sequences substantially identical thereto, can be represented in thetraditional single character format (See the inside back cover ofStryer, Lubert. Biochemistry, 3rd Ed., W.H. Freeman & Co., New York.) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified elsewhere in this patentspecification are particularly contemplated for use in this aspect ofthe invention. Protein and/or nucleic acid sequence homologies may beevaluated using any of the variety of sequence comparison algorithms andprograms known in the art. Such algorithms and programs include, but areby no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988;Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al.,Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., MethodsEnzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol.215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project. Atleast twenty-one other genomes have already been sequenced, including,for example, M. genitalium (Fraser et al., 1995), M. jannaschii (Bult etal., 1996), H. influenzae (Fleischmann et al., 1995), E. coli (Blattneret al., 1997) and yeast (S. cerevisiae) (Mewes et al., 1997) and D.melanogaster (Adams et al., 2000). Significant progress has also beenmade in sequencing the genomes of model organism, such as mouse, C.elegans and Arabadopsis sp. Several databases containing genomicinformation annotated with some functional information are maintained bydifferent organization and are accessible via the internet.

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402,1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more preferably less thanabout 0.01 and most preferably less than about 0.001.

In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet etal., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also beused (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, a nucleic acid or polypeptide sequence ofthe invention can be stored, recorded, and manipulated on any mediumwhich can be read and accessed by a computer.

Accordingly, the invention provides computers, computer systems,computer readable mediums, computer programs products and the likerecorded or stored thereon the nucleic acid and polypeptide sequences ofthe invention. As used herein, the words “recorded” and “stored” referto a process for storing information on a computer medium. A skilledartisan can readily adopt any known methods for recording information ona computer readable medium to generate manufactures comprising one ormore of the nucleic acid and/or polypeptide sequences of the invention.

The polypeptides of the invention include the polypeptide sequences ofGroup B amino acid sequences, the exemplary sequences of the invention,and sequences substantially identical thereto, and fragments of any ofthe preceding sequences. Substantially identical, or homologous,polypeptide sequences refer to a polypeptide sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to an exemplary sequence of the invention, e.g., apolypeptide sequences of the Group B amino acid sequences.

Homology may be determined using any of the computer programs andparameters described herein, including FASTA version 3.0t78 with thedefault parameters or with any modified parameters. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. The polypeptidefragments comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutiveamino acids of the polypeptides of Group B amino acid sequences andsequences substantially identical thereto. It will be appreciated thatthe polypeptide codes as set forth in Group B amino acid sequences andsequences substantially identical thereto, can be represented in thetraditional single character format or three letter format (See theinside back cover of Stryer, Lubert. Biochemistry, 3rd Ed., W.H. Freeman& Co., New York.) or in any other format which relates the identity ofthe polypeptides in a sequence.

A nucleic acid or polypeptide sequence of the invention can be stored,recorded and manipulated on any medium which can be read and accessed bya computer. As used herein, the words “recorded” and “stored” refer to aprocess for storing information on a computer medium. A skilled artisancan readily adopt any of the presently known methods for recordinginformation on a computer readable medium to generate manufacturescomprising one or more of the nucleic acid sequences as set forth inGroup A nucleic acid sequences and sequences substantially identicalthereto, one or more of the polypeptide sequences as set forth in GroupB amino acid sequences and sequences substantially identical thereto.Another aspect of the invention is a computer readable medium havingrecorded thereon at least 2, 5, 10, 15, or 20 or more nucleic acidsequences as set forth in Group A nucleic acid sequences and sequencessubstantially identical thereto.

Another aspect of the invention is a computer readable medium havingrecorded thereon one or more of the nucleic acid sequences as set forthin Group A nucleic acid sequences and sequences substantially identicalthereto. Another aspect of the invention is a computer readable mediumhaving recorded thereon one or more of the polypeptide sequences as setforth in Group B amino acid sequences and sequences substantiallyidentical thereto. Another aspect of the invention is a computerreadable medium having recorded thereon at least 2, 5, 10, 15, or 20 ormore of the sequences as set forth above.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems which store and manipulate the sequenceinformation described herein. One example of a computer system 100 isillustrated in block diagram form in FIG. 1. As used herein, “a computersystem” refers to the hardware components, software components and datastorage components used to analyze a nucleotide sequence of a nucleicacid sequence as set forth in Group A nucleic acid sequences andsequences substantially identical thereto, or a polypeptide sequence asset forth in the Group B amino acid sequences. The computer system 100typically includes a processor for processing, accessing andmanipulating the sequence data. The processor 105 can be any well-knowntype of central processing unit, such as, for example, the Pentium IIIfrom Intel Corporation, or similar processor from Sun, Motorola, Compaq,AMD or International Business Machines.

Typically the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular aspect, the computer system 100 includes a processor105 connected to a bus which is connected to a main memory 115(preferably implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some aspects, the computer system 100further includes one or more data retrieving device 118 for reading thedata stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some aspects, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of anucleic acid sequence as set forth in Group A nucleic acid sequences andsequences substantially identical thereto, or a polypeptide sequence asset forth in Group B amino acid sequences and sequences substantiallyidentical thereto, (such as search tools, compare tools and modelingtools etc.) may reside in main memory 115 during execution.

In some aspects, the computer system 100 may further comprise a sequencecomparison algorithm for comparing a nucleic acid sequence as set forthin Group A nucleic acid sequences and sequences substantially identicalthereto, or a polypeptide sequence as set forth in Group B amino acidsequences and sequences substantially identical thereto, stored on acomputer readable medium to a reference nucleotide or polypeptidesequence(s) stored on a computer readable medium. A “sequence comparisonalgorithm” refers to one or more programs which are implemented (locallyor remotely) on the computer system 100 to compare a nucleotide sequencewith other nucleotide sequences and/or compounds stored within a datastorage means. For example, the sequence comparison algorithm maycompare the nucleotide sequences of a nucleic acid sequence as set forthin Group A nucleic acid sequences and sequences substantially identicalthereto, or a polypeptide sequence as set forth in Group B amino acidsequences and sequences substantially identical thereto, stored on acomputer readable medium to reference sequences stored on a computerreadable medium to identify homologies or structural motifs.

FIG. 2 is a flow diagram illustrating one aspect of a process 200 forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence as set forth in Group A nucleic acid sequences and sequencessubstantially identical thereto, or a polypeptide sequence as set forthin Group B amino acid sequences and sequences substantially identicalthereto, a data storage device having retrievably stored thereonreference nucleotide sequences or polypeptide sequences to be comparedto a nucleic acid sequence as set forth in Group A nucleic acidsequences and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences andsequences substantially identical thereto and a sequence comparer forconducting the comparison. The sequence comparer may indicate a homologylevel between the sequences compared or identify structural motifs inthe above described nucleic acid code of Group A nucleic acid sequencesand sequences substantially identical thereto, or a polypeptide sequenceas set forth in Group B amino acid sequences and sequences substantiallyidentical thereto, or it may identify structural motifs in sequenceswhich are compared to these nucleic acid codes and polypeptide codes. Insome aspects, the data storage device may have stored thereon thesequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of thenucleic acid sequences as set forth in Group A nucleic acid sequencesand sequences substantially identical thereto, or the polypeptidesequences as set forth in Group B amino acid sequences and sequencessubstantially identical thereto.

Another aspect of the invention is a method for determining the level ofhomology between a nucleic acid sequence as set forth in Group A nucleicacid sequences and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences andsequences substantially identical thereto and a reference nucleotidesequence. The method including reading the nucleic acid code or thepolypeptide code and the reference nucleotide or polypeptide sequencethrough the use of a computer program which determines homology levelsand determining homology between the nucleic acid code or polypeptidecode and the reference nucleotide or polypeptide sequence with thecomputer program. The computer program may be any of a number ofcomputer programs for determining homology levels, including thosespecifically enumerated herein, (e.g., BLAST2N with the defaultparameters or with any modified parameters). The method may beimplemented using the computer systems described above. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 ormore of the above described nucleic acid sequences as set forth in theGroup A nucleic acid sequences, or the polypeptide sequences as setforth in the Group B amino acid sequences through use of the computerprogram and determining homology between the nucleic acid codes orpolypeptide codes and reference nucleotide sequences or polypeptidesequences.

FIG. 3 is a flow diagram illustrating one aspect of a process 250 in acomputer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is preferably in the single letter amino acidcode so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of a nucleic acid sequence as setforth in the invention, to one or more reference nucleotide sequences inorder to determine whether the nucleic acid code of Group A nucleic acidsequences and sequences substantially identical thereto, differs from areference nucleic acid sequence at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or a nucleic acid sequence as set forth inGroup A nucleic acid sequences and sequences substantially identicalthereto. In one aspect, the computer program may be a program whichdetermines whether a nucleic acid sequence as set forth in Group Anucleic acid sequences and sequences substantially identical thereto,contains a single nucleotide polymorphism (SNP) with respect to areference nucleotide sequence.

Accordingly, another aspect of the invention is a method for determiningwhether a nucleic acid sequence as set forth in Group A nucleic acidsequences and sequences substantially identical thereto, differs at oneor more nucleotides from a reference nucleotide sequence comprising thesteps of reading the nucleic acid code and the reference nucleotidesequence through use of a computer program which identifies differencesbetween nucleic acid sequences and identifying differences between thenucleic acid code and the reference nucleotide sequence with thecomputer program. In some aspects, the computer program is a programwhich identifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic acidsequences as set forth in Group A nucleic acid sequences and sequencessubstantially identical thereto and the reference nucleotide sequencesthrough the use of the computer program and identifying differencesbetween the nucleic acid codes and the reference nucleotide sequenceswith the computer program.

In other aspects the computer based system may further comprise anidentifier for identifying features within a nucleic acid sequence asset forth in the Group A nucleic acid sequences or a polypeptidesequence as set forth in Group B amino acid sequences and sequencessubstantially identical thereto.

An “identifier” refers to one or more programs which identifies certainfeatures within a nucleic acid sequence as set forth in Group A nucleicacid sequences and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences andsequences substantially identical thereto. In one aspect, the identifiermay comprise a program which identifies an open reading frame in anucleic acid sequence as set forth in Group A nucleic acid sequences andsequences substantially identical thereto.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group. Alternatively, the features may bestructural polypeptide motifs such as alpha helices, beta sheets, orfunctional polypeptide motifs such as enzymatic active sites,helix-turn-helix motifs or other motifs known to those skilled in theart.

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence. It should be noted, that if thefeature attribute is not found in the first sequence at the decisionstate 316, the process 300 moves directly to the decision state 320 inorder to determine if any more features exist in the database.

Accordingly, another aspect of the invention is a method of identifyinga feature within a nucleic acid sequence as set forth in Group A nucleicacid sequences and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences andsequences substantially identical thereto, comprising reading thenucleic acid code(s) or polypeptide code(s) through the use of acomputer program which identifies features therein and identifyingfeatures within the nucleic acid code(s) with the computer program. Inone aspect, computer program comprises a computer program whichidentifies open reading frames. The method may be performed by reading asingle sequence or at least 2, 5, 10, 15, 20, 25, 30, or 40 of thenucleic acid sequences as set forth in Group A nucleic acid sequencesand sequences substantially identical thereto, or the polypeptidesequences as set forth in Group B amino acid sequences and sequencessubstantially identical thereto, through the use of the computer programand identifying features within the nucleic acid codes or polypeptidecodes with the computer program.

A nucleic acid sequence as set forth in Group A nucleic acid sequencesand sequences substantially identical thereto, or a polypeptide sequenceas set forth in Group B amino acid sequences and sequences substantiallyidentical thereto, may be stored and manipulated in a variety of dataprocessor programs in a variety of formats. For example, a nucleic acidsequence as set forth in Group A nucleic acid sequences and sequencessubstantially identical thereto, or a polypeptide sequence as set forthin Group B amino acid sequences and sequences substantially identicalthereto, may be stored as text in a word processing file, such asMicrosoft WORD™ or WORDPERFECT™ or as an ASCII file in a variety ofdatabase programs familiar to those of skill in the art, such as DB2™,SYBASE™, or ORACLE™. In addition, many computer programs and databasesmay be used as sequence comparison algorithms, identifiers, or sourcesof reference nucleotide sequences or polypeptide sequences to becompared to a nucleic acid sequence as set forth in Group A nucleic acidsequences and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences andsequences substantially identical thereto. The following list isintended not to limit the invention but to provide guidance to programsand databases which are useful with the nucleic acid sequences as setforth in Group A nucleic acid sequences and sequences substantiallyidentical thereto, or the polypeptide sequences as set forth in Group Bamino acid sequences and sequences substantially identical thereto.

The programs and databases which may be used include, but are notlimited to: MacPattern (EMBL), DiscoveryBase (Molecular ApplicationsGroup), GeneMine (Molecular Applications Group), Look (MolecularApplications Group), MacLook (Molecular Applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215:403,1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHARNm (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.),WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug-Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwents'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to an exemplary sequence of theinvention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313,SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ IDNO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341,SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ IDNO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369,SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377 or SEQ IDNO:379). The stringent conditions can be highly stringent conditions,medium stringent conditions and/or low stringent conditions, includingthe high and reduced stringency conditions described herein. In oneaspect, it is the stringency of the wash conditions that set forth theconditions which determine whether a nucleic acid is within the scope ofthe invention, as discussed below.

In alternative aspects, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA (single or double stranded),antisense or sequences encoding antibody binding peptides (epitopes),motifs, active sites and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/mlsheared and denatured salmon sperm DNA). In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency conditions comprising 35% formamide at a reduced temperatureof 35° C.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent) and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4−9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at T_(m)-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

All of the foregoing hybridizations would be considered to be underconditions of high stringency.

Following hybridization, a filter can be washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content) and the nucleic acid type (e.g., RNA v. DNA). Examples ofprogressively higher stringency condition washes are as follows: 2×SSC,0.1% SDS at room temperature for 15 minutes (low stringency); 0.1×SSC,0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes. Some other examples are givenbelow.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention. Forexample, the preceding methods may be used to isolate nucleic acidshaving a sequence with at least about 97%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a nucleic acidsequence selected from the group consisting of one of the sequences ofGroup A nucleic acid sequences and sequences substantially identicalthereto, or fragments comprising at least about 10, 15, 20, 25, 30, 35,40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases thereofand the sequences complementary thereto. Homology may be measured usingthe alignment algorithm. For example, the homologous polynucleotides mayhave a coding sequence which is a naturally occurring allelic variant ofone of the coding sequences described herein. Such allelic variants mayhave a substitution, deletion or addition of one or more nucleotideswhen compared to the nucleic acids of Group A nucleic acid sequences orthe sequences complementary thereto.

Additionally, the above procedures may be used to isolate nucleic acidswhich encode polypeptides having at least about 99%, 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a polypeptidehaving the sequence of one of Group B amino acid sequences and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof as determined using a sequence alignment algorithm (e.g., suchas the FASTA version 3.0t78 algorithm with the default parameters).

Oligonucleotides Probes and Methods for Using Them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with a xylanaseactivity or fragments thereof or for identifying xylanase genes. In oneaspect, the probe comprises at least 10 consecutive bases of a nucleicacid of the invention. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutivebases of a sequence as set forth in a nucleic acid of the invention. Theprobes identify a nucleic acid by binding and/or hybridization. Theprobes can be used in arrays of the invention, see discussion below,including, e.g., capillary arrays. The probes of the invention can alsobe used to isolate other nucleic acids or polypeptides.

The isolated nucleic acids of Group A nucleic acid sequences andsequences substantially identical thereto, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of Group A nucleic acid sequences and sequences substantiallyidentical thereto, or the sequences complementary thereto may also beused as probes to determine whether a biological sample, such as a soilsample, contains an organism having a nucleic acid sequence of theinvention or an organism from which the nucleic acid was obtained. Insuch procedures, a biological sample potentially harboring the organismfrom which the nucleic acid was isolated is obtained and nucleic acidsare obtained from the sample. The nucleic acids are contacted with theprobe under conditions which permit the probe to specifically hybridizeto any complementary sequences from which are present therein.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley 503 Sons, Inc. (1997) and Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor LaboratoryPress (1989.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook,supra. Alternatively, the amplification may comprise a ligase chainreaction, 3SR, or strand displacement reaction. (See Barany, F., “TheLigase Chain Reaction in a PCR World”, PCR Methods and Applications1:5-16, 1991; E. Fahy et al., “Self-sustained Sequence Replication(3SR): An Isothermal Transcription-based Amplification SystemAlternative to PCR”, PCR Methods and Applications 1:25-33, 1991; andWalker G. T. et al., “Strand Displacement Amplification-an Isothermal invitro DNA Amplification Technique”, Nucleic Acid Research 20:1691-1696,1992). In such procedures, the nucleic acids in the sample are contactedwith the probes, the amplification reaction is performed and anyresulting amplification product is detected. The amplification productmay be detected by performing gel electrophoresis on the reactionproducts and staining the gel with an intercalator such as ethidiumbromide. Alternatively, one or more of the probes may be labeled with aradioactive isotope and the presence of a radioactive amplificationproduct may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of the sequences of Group Anucleic acid sequences and sequences substantially identical thereto,may also be used in chromosome walking procedures to identify clonescontaining genomic sequences located adjacent to the sequences of GroupA nucleic acid sequences and sequences substantially identical thereto.Such methods allow the isolation of genes which encode additionalproteins from the host organism.

The isolated nucleic acids of Group A nucleic acid sequences andsequences substantially identical thereto, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of Group A nucleic acid sequences and sequences substantiallyidentical thereto, or the sequences complementary thereto may be used asprobes to identify and isolate related nucleic acids. In some aspects,the related nucleic acids may be cDNAs or genomic DNAs from organismsother than the one from which the nucleic acid was isolated. Forexample, the other organisms may be related organisms. In suchprocedures, a nucleic acid sample is contacted with the probe underconditions which permit the probe to specifically hybridize to relatedsequences. Hybridization of the probe to nucleic acids from the relatedorganism is then detected using any of the methods described above.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula: T_(m)=81.5+16.6(log[Na⁺])+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA, 50%formamide. The formulas for SSC and Denhardt's solutions are listed inSambrook et al., supra.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the T_(m). Forshorter probes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T_(m). Typically, for hybridizations in6×SSC, the hybridization is conducted at approximately 68° C. Usually,for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C.

Inhibiting Expression of Xylanases

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acids of the invention, e.g.,xylanase-encoding nucleic acids. Antisense sequences are capable ofinhibiting the transport, splicing or transcription of xylanase-encodinggenes. The inhibition can be effected through the targeting of genomicDNA or messenger RNA. The transcription or function of targeted nucleicacid can be inhibited, for example, by hybridization and/or cleavage.One particularly useful set of inhibitors provided by the presentinvention includes oligonucleotides which are able to either bindxylanase gene or message, in either case preventing or inhibiting theproduction or function of xylanase. The association can be throughsequence specific hybridization. Another useful class of inhibitorsincludes oligonucleotides which cause inactivation or cleavage ofxylanase message. The oligonucleotide can have enzyme activity whichcauses such cleavage, such as ribozymes. The oligonucleotide can bechemically modified or conjugated to an enzyme or composition capable ofcleaving the complementary nucleic acid. A pool of many different sucholigonucleotides can be screened for those with the desired activity.Thus, the invention provides various compositions for the inhibition ofxylanase expression on a nucleic acid and/or protein level, e.g.,antisense, iRNA and ribozymes comprising xylanase sequences of theinvention and the anti-xylanase antibodies of the invention.

Inhibition of xylanase expression can have a variety of industrialapplications. For example, inhibition of xylanase expression can slow orprevent spoilage. Spoilage can occur when polysaccharides, e.g.,structural polysaccharides, are enzymatically degraded. This can lead tothe deterioration, or rot, of fruits and vegetables. In one aspect, useof compositions of the invention that inhibit the expression and/oractivity of xylanases, e.g., antibodies, antisense oligonucleotides,ribozymes and RNAi, are used to slow or prevent spoilage. Thus, in oneaspect, the invention provides methods and compositions comprisingapplication onto a plant or plant product (e.g., a cereal, a grain, afruit, seed, root, leaf, etc.) antibodies, antisense oligonucleotides,ribozymes and RNAi of the invention to slow or prevent spoilage. Thesecompositions also can be expressed by the plant (e.g., a transgenicplant) or another organism (e.g., a bacterium or other microorganismtransformed with a xylanase gene of the invention).

The compositions of the invention for the inhibition of xylanaseexpression (e.g., antisense, iRNA, ribozymes, antibodies) can be used aspharmaceutical compositions, e.g., as anti-pathogen agents or in othertherapies, e.g., as anti-microbials for, e.g., Salmonella.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingxylanase message which can inhibit xylan hydrolase activity (e.g.,catalyzing hydrolysis of internal β-1,4-xylosidic linkages) by targetingmRNA. Strategies for designing antisense oligonucleotides are welldescribed in the scientific and patent literature, and the skilledartisan can design such xylanase oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl)glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensexylanase sequences of the invention (see, e.g., Gold (1995) J. of Biol.Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding xylanase message.These ribozymes can inhibit xylanase activity by, e.g., targeting mRNA.Strategies for designing ribozymes and selecting the xylanase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it canbe released from that RNA to bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNascP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a xylanase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of a xylanase gene. In oneaspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length. While the invention is not limited byany particular mechanism of action, the RNAi can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a xylanase. These methodscan be repeated or used in various combinations to generate xylanaseshaving an altered or different activity or an altered or differentstability from that of a xylanase encoded by the template nucleic acid.These methods also can be repeated or used in various combinations,e.g., to generate variations in gene/message expression, messagetranslation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly (e.g.,GeneReassembly™, see, e.g., U.S. Pat. No. 6,537,776), gene sitesaturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor(1985) “The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor(1985) “The rapid generation of oligonucleotide-directed mutations athigh frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13:8765-8787 (1985); Nakamaye (1986) “Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis” Nucl. Acids Res.14: 9679-9698; Sayers (1988) “Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; andSayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer (1988) “Improved enzymatic invitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. Nos. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLE CELLSAND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre etal., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate xylanases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high or lowtemperatures, and the like). Polypeptides encoded by the modifiednucleic acids can be screened for an activity before testing for xylanhydrolysis or other activity. Any testing modality or protocol can beused, e.g., using a capillary array platform. See, e.g., U.S. Pat. Nos.6,361,974; 6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., axylanase or an antibody of the invention, so as to generate a set ofprogeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, optionally, asecond homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprised of, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position X 100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,xylanases) molecules such that all 20 natural amino acids arerepresented at the one specific amino acid position corresponding to thecodon position mutagenized in the parental polynucleotide (other aspectsuse less than all 20 natural combinations). The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g. cloned into asuitable host, e.g., E. Coli host, using, e.g., an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedxylan hydrolysis activity under alkaline or acidic conditions), it canbe sequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (gene site saturated mutagenesis (GSSM™)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence and preferably but not necessarilya second homologous sequence. The downstream progeny translationalproducts from the use of such oligos include all possible amino acidchanges at each amino acid site along the polypeptide, because thedegeneracy of the N,N,N sequence includes codons for all 20 amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,N cassette) is used for subjecting each original codon in a parentalpolynucleotide template to a full range of codon substitutions. Inanother aspect, at least two degenerate N,N,N cassettes are used—eitherin the same oligo or not, for subjecting at least two original codons ina parental polynucleotide template to a full range of codonsubstitutions. Thus, more than one N,N,N sequence can be contained inone oligo to introduce amino acid mutations at more than one site. Thisplurality of N,N,N sequences can be directly contiguous, or separated byone or more additional nucleotide sequence(s). In another aspect, oligosserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,N sequence, tointroduce any combination or permutation of amino acid additions,deletions and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N)_(n)sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g. in an oligo)a degenerate triplet sequence comprised of only one N, where the N canbe in the first second or third position of the triplet. Any other basesincluding any combinations and permutations thereof can be used in theremaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides a means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changesand point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in one aspect of this invention, each saturation mutagenesisreaction vessel contains polynucleotides encoding at least 20 progenypolypeptide molecules such that all 20 amino acids are represented atthe one specific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is preferably everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(preferably a subset totaling from 15 to 100,000) to mutagenesis.Preferably, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations are preferablyintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Exemplary cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette ispreferably about 1-500 bases in length) in defined polynucleotidesequence to be mutagenized (wherein the sequence to be mutagenized ispreferably from about 15 to 100,000 bases in length). Thus, a group ofmutations (ranging from 1 to 100 mutations) is introduced into eachcassette to be mutagenized. A grouping of mutations to be introducedinto one cassette can be different or the same from a second grouping ofmutations to be introduced into a second cassette during the applicationof one round of saturation mutagenesis. Such groupings are exemplifiedby deletions, additions, groupings of particular codons and groupings ofparticular nucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF) and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one exemplification a grouping of mutations that can be introducedinto a mutagenic cassette, this invention specifically provides fordegenerate codon substitutions (using degenerate oligos) that code for2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20amino acids at each position and a library of polypeptides encodedthereby.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., xylanases or antibodies of theinvention, with new or altered properties.

SLR is a method of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See, e.g., U.S. patent application Ser. No.09/332,835 entitled “Synthetic Ligation Reassembly in DirectedEvolution” and filed on Jun. 14, 1999 (“U.S. Ser. No. 09/332,835”). Inone aspect, SLR comprises the following steps: (a) providing a templatepolynucleotide, wherein the template polynucleotide comprises sequenceencoding a homologous gene; (b) providing a plurality of building blockpolynucleotides, wherein the building block polynucleotides are designedto cross-over reassemble with the template polynucleotide at apredetermined sequence, and a building block polynucleotide comprises asequence that is a variant of the homologous gene and a sequencehomologous to the template polynucleotide flanking the variant sequence;(c) combining a building block polynucleotide with a templatepolynucleotide such that the building block polynucleotide cross-overreassembles with the template polynucleotide to generate polynucleotidescomprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

Synthetic Gene Reassembly

In one aspect, the present invention provides a non-stochastic methodtermed synthetic gene reassembly (e.g., GeneReassembly™, see, e.g., U.S.Pat. No. 6,537,776), which differs from stochastic shuffling in that thenucleic acid building blocks are not shuffled or concatenated orchimerized randomly, but rather are assembled non-stochastically.

The synthetic gene reassembly method does not depend on the presence ofa high level of homology between polynucleotides to be shuffled. Theinvention can be used to non-stochastically generate libraries (or sets)of progeny molecules comprised of over 10¹⁰⁰ different chimeras.Conceivably, synthetic gene reassembly can even be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprised of the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

In one aspect, synthetic gene reassembly comprises a method of: 1)preparing a progeny generation of molecule(s) (including a moleculecomprising a polynucleotide sequence, e.g., a molecule comprising apolypeptide coding sequence), that is mutagenized to achieve at leastone point mutation, addition, deletion, &/or chimerization, from one ormore ancestral or parental generation template(s); 2) screening theprogeny generation molecule(s), e.g., using a high throughput method,for at least one property of interest (such as an improvement in anenzyme activity); 3) optionally obtaining &/or cataloguing structural&/or and functional information regarding the parental &/or progenygeneration molecules; and 4) optionally repeating any of steps 1) to 3).In one aspect, there is generated (e.g., from a parent polynucleotidetemplate), in what is termed “codon site-saturation mutagenesis,” aprogeny generation of polynucleotides, each having at least one set ofup to three contiguous point mutations (i.e. different bases comprisinga new codon), such that every codon (or every family of degeneratecodons encoding the same amino acid) is represented at each codonposition. Corresponding to, and encoded by, this progeny generation ofpolynucleotides, there is also generated a set of progeny polypeptides,each having at least one single amino acid point mutation. In a oneaspect, there is generated, in what is termed “amino acidsite-saturation mutagenesis”, one such mutant polypeptide for each ofthe 19 naturally encoded polypeptide-forming alpha-amino acidsubstitutions at each and every amino acid position along thepolypeptide. This yields, for each and every amino acid position alongthe parental polypeptide, a total of 20 distinct progeny polypeptidesincluding the original amino acid, or potentially more than 21 distinctprogeny polypeptides if additional amino acids are used either insteadof or in addition to the 20 naturally encoded amino acids.

Thus, in another aspect, this approach is also serviceable forgenerating mutants containing, in addition to &/or in combination withthe 20 naturally encoded polypeptide-forming alpha-amino acids, otherrare &/or not naturally-encoded amino acids and amino acid derivatives.In yet another aspect, this approach is also serviceable for generatingmutants by the use of, in addition to &/or in combination with naturalor unaltered codon recognition systems of suitable hosts, altered,mutagenized, &/or designer codon recognition systems (such as in a hostcell with one or more altered tRNA molecules.

In yet another aspect, this invention relates to recombination and morespecifically to a method for preparing polynucleotides encoding apolypeptide by a method of in vivo re-assortment of polynucleotidesequences containing regions of partial homology, assembling thepolynucleotides to form at least one polynucleotide and screening thepolynucleotides for the production of polypeptide(s) having a usefulproperty.

In yet another aspect, this invention is serviceable for analyzing andcataloguing, with respect to any molecular property (e.g. an enzymaticactivity) or combination of properties allowed by current technology,the effects of any mutational change achieved (including particularlysaturation mutagenesis). Thus, a comprehensive method is provided fordetermining the effect of changing each amino acid in a parentalpolypeptide into each of at least 19 possible substitutions. This allowseach amino acid in a parental polypeptide to be characterized andcatalogued according to its spectrum of potential effects on ameasurable property of the polypeptide.

In one aspect, an intron may be introduced into a chimeric progenymolecule by way of a nucleic acid building block. Introns often haveconsensus sequences at both termini in order to render them operational.In addition to enabling gene splicing, introns may serve an additionalpurpose by providing sites of homology to other nucleic acids to enablehomologous recombination. For this purpose, and potentially others, itmay be sometimes desirable to generate a large nucleic acid buildingblock for introducing an intron. If the size is overly large easilygenerating by direct chemical synthesis of two single stranded oligos,such a specialized nucleic acid building block may also be generated bydirect chemical synthesis of more than two single stranded oligos or byusing a polymerase-based amplification reaction.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In a one aspect of the invention, the annealedbuilding pieces are treated with an enzyme, such as a ligase (e.g., T4DNA ligase) to achieve covalent bonding of the building pieces.

Coupling can occur in a manner that does not make use of everynucleotide in a participating overhang. The coupling is particularlylively to survive (e.g. in a transformed host) if the couplingreinforced by treatment with a ligase enzyme to form what may bereferred to as a “gap ligation” or a “gapped ligation”. This type ofcoupling can contribute to generation of unwanted background product(s),but it can also be used advantageously increase the diversity of theprogeny library generated by the designed ligation reassembly. Certainoverhangs are able to undergo self-coupling to form a palindromiccoupling. A coupling is strengthened substantially if it is reinforcedby treatment with a ligase enzyme. Lack of 5′ phosphates on theseoverhangs can be used advantageously to prevent this type of palindromicself-ligation. Accordingly, this invention provides that nucleic acidbuilding blocks can be chemically made (or ordered) that lack a 5′phosphate group. Alternatively, they can be removed, e.g. by treatmentwith a phosphatase enzyme, such as a calf intestinal alkalinephosphatase (CIAP), in order to prevent palindromic self-ligations inligation reassembly processes.

In a another aspect, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes. Thexylanases of the present invention can be mutagenized in accordance withthe methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates (e.g., polynucleotides ofGroup A nucleic acid sequences) are aligned in order to select one ormore demarcation points, which demarcation points can be located at anarea of homology. The demarcation points can be used to delineate theboundaries of nucleic acid building blocks to be generated. Thus, thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the progenymolecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates and preferably at almost all of theprogenitor templates. Even more preferably still a serviceabledemarcation point is an area of homology that is shared by all of theprogenitor templates.

In a one aspect, the gene reassembly process is performed exhaustivelyin order to generate an exhaustive library. In other words, all possibleordered combinations of the nucleic acid building blocks are representedin the set of finalized chimeric nucleic acid molecules. At the sametime, the assembly order (i.e. the order of assembly of each buildingblock in the 5′ to 3 sequence of each finalized chimeric nucleic acid)in each combination is by design (or non-stochastic). Because of thenon-stochastic nature of the method, the possibility of unwanted sideproducts is greatly reduced.

In another aspect, the method provides that the gene reassembly processis performed systematically, for example to generate a systematicallycompartmentalized library, with compartments that can be screenedsystematically, e.g., one by one. In other words the invention providesthat, through the selective and judicious use of specific nucleic acidbuilding blocks, coupled with the selective and judicious use ofsequentially stepped assembly reactions, an experimental design can beachieved where specific sets of progeny products are made in each ofseveral reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated preferably comprise a library of finalizedchimeric nucleic acid molecules having an overall assembly order that ischosen by design. In a particularly aspect, such a generated library iscomprised of greater than 10³ to greater than 10¹⁰⁰⁰ different progenymolecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one aspect, this polynucleotide is a gene,which may be a man-made gene. According to another aspect, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g., by mutagenesis) or in an in vivoprocess (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another aspect, the invention provides that a nucleicacid building block can be used to introduce an intron. Thus, theinvention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). Preferably, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In a one aspect,the recombination is facilitated by, or occurs at, areas of homologybetween the man-made, intron-containing gene and a nucleic acid, whichserves as a recombination partner. In one aspect, the recombinationpartner may also be a nucleic acid generated by the invention, includinga man-made gene or a man-made gene pathway. Recombination may befacilitated by or may occur at areas of homology that exist at the one(or more) artificially introduced intron(s) in the man-made gene.

The synthetic gene reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which preferably hastwo ligatable ends. The two ligatable ends on each nucleic acid buildingblock may be two blunt ends (i.e. each having an overhang of zeronucleotides), or preferably one blunt end and one overhang, or morepreferably still two overhangs.

A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

In one aspect, a nucleic acid building block is generated by chemicalsynthesis of two single-stranded nucleic acids (also referred to assingle-stranded oligos) and contacting them so as to allow them toanneal to form a double-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Exemplarysizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Otherexemplary size ranges are also provided, which have lower limits of from1 bp to 10,000 bp (including every integer value in between) and upperlimits of from 2 bp to 100,000 bp (including every integer value inbetween).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one aspect, a double-stranded nucleic acid building blockis generated by first generating two single stranded nucleic acids andallowing them to anneal to form a double-stranded nucleic acid buildingblock. The two strands of a double-stranded nucleic acid building blockmay be complementary at every nucleotide apart from any that form anoverhang; thus containing no mismatches, apart from any overhang(s).According to another aspect, the two strands of a double-strandednucleic acid building block are complementary at fewer than everynucleotide apart from any that form an overhang. Thus, according to thisaspect, a double-stranded nucleic acid building block can be used tointroduce codon degeneracy. Preferably the codon degeneracy isintroduced using the site-saturation mutagenesis described herein, usingone or more N,N,G/T cassettes or alternatively using one or more N,N,Ncassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect the invention described herein is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionwhich allow for the directed molecular evolution of highly complexlinear sequences, such as DNA, RNA or proteins thorough recombination.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,xylanases or antibodies of the invention, with new or alteredproperties. Optimized directed evolution is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionthat allow for the directed molecular evolution of nucleic acids throughrecombination. Optimized directed evolution allows generation of a largepopulation of evolved chimeric sequences, wherein the generatedpopulation is significantly enriched for sequences that have apredetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLA™ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new xylanase phenotype is identified, re-isolated,again modified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including, e.g., xylanase activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new xylanase phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,xylanases, and the like. In vivo shuffling can be performed utilizingthe natural property of cells to recombine multimers. Whilerecombination in vivo has provided the major natural route to moleculardiversity, genetic recombination remains a relatively complex processthat involves 1) the recognition of homologies; 2) strand cleavage,strand invasion, and metabolic steps leading to the production ofrecombinant chiasma; and finally 3) the resolution of chiasma intodiscrete recombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In another aspect, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257 andcombinations thereof) into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

Therefore, in another aspect of the invention, novel polynucleotides canbe generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   a) Primers that include a poly-A head and poly-T tail which when        made single-stranded would provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNAseH.    -   b) Primers that include unique restriction cleavage sites can be        utilized. Multiple sites, a battery of unique sequences and        repeated synthesis and ligation steps would be required.    -   c) The inner few bases of the primer could be thiolated and an        exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by:

-   1) The use of vectors only stably maintained when the construct is    reduced in complexity.-   2) The physical recovery of shortened vectors by physical    procedures. In this case, the cloning vector would be recovered    using standard plasmid isolation procedures and size fractionated on    either an agarose gel, or column with a low molecular weight cut off    utilizing standard procedures.-   3) The recovery of vectors containing interrupted genes which can be    selected when insert size decreases.-   4) The use of direct selection techniques with an expression vector    and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates a method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are described. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence. The quasi-repeatedsequences are separately or collectively amplified and ligated intorandom assemblies such that all possible permutations and combinationsare available in the population of ligated molecules. The number ofquasi-repeat units can be controlled by the assembly conditions. Theaverage number of quasi-repeated units in a construct is defined as therepetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector and transfected into an appropriate host cell. The cells are thenpropagated and “reductive reassortment” is effected. The rate of thereductive reassortment process may be stimulated by the introduction ofDNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, virion, or otherpredetermined compound or structure.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (e.g., catalysts,solutes for increasing osmolarity of an aqueous solution and the like)and/or can be subjected to one or more additional cycles of shufflingand/or selection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N-3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[α]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[α]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-j]-quinoline(“N-hydroxy-IQ”) andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-j]-pyridine(“N-hydroxy-PhIP”). Exemplary means for slowing or halting PCRamplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N-3-Adenine). Particularly encompassed means are DNAadducts or polynucleotides comprising the DNA adducts from thepolynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

In another aspect the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., xylanase) sequences of theinvention. The invention also provides additional methods for isolatingxylanases using the nucleic acids and polypeptides of the invention. Inone aspect, the invention provides for variants of a xylanase codingsequence (e.g., a gene, cDNA or message) of the invention, which can bealtered by any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids areevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations”.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in Arkin, A. P. and Youvan, D. C., PNAS, USA,89:7811-7815, 1992.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described inDelegrave, S. and Youvan, D. C., Biotechnology Research, 11:1548-1552,1993. Random and site-directed mutagenesis are described in Arnold, F.H., Current Opinion in Biotechnology, 4:450-455, 1993.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis” and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis.

The variants of the polypeptides of Group B amino acid sequences may bevariants in which one or more of the amino acid residues of thepolypeptides of the Group B amino acid sequences are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Alanine,Valine, Leucine and Isoleucine with another aliphatic amino acid;replacement of a Serine with a Threonine or vice versa; replacement ofan acidic residue such as Aspartic acid and Glutamic acid with anotheracidic residue; replacement of a residue bearing an amide group, such asAsparagine and Glutamine, with another residue bearing an amide group;exchange of a basic residue such as Lysine and Arginine with anotherbasic residue; and replacement of an aromatic residue such asPhenylalanine, Tyrosine with another aromatic residue.

Other variants are those in which one or more of the amino acid residuesof the polypeptides of the Group B amino acid sequences includes asubstituent group.

Still other variants are those in which the polypeptide is associatedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol).

Additional variants are those in which additional amino acids are fusedto the polypeptide, such as a leader sequence, a secretory sequence, aproprotein sequence or a sequence which facilitates purification,enrichment, or stabilization of the polypeptide.

In some aspects, the fragments, derivatives and analogs retain the samebiological function or activity as the polypeptides of Group B aminoacid sequences and sequences substantially identical thereto. In otheraspects, the fragment, derivative, or analog includes a proprotein, suchthat the fragment, derivative, or analog can be activated by cleavage ofthe proprotein portion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying xylanase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding a xylanase toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding a xylanase modified to increase itsexpression in a host cell, xylanase so modified, and methods of makingthe modified xylanases. The method comprises identifying a“non-preferred” or a “less preferred” codon in xylanase-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asStreptomyces diversa, Lactobacillus gasseri, Lactococcus lactis,Lactococcus cremoris, Bacillus subtilis. Exemplary host cells alsoinclude eukaryotic organisms, e.g., various yeast, such as Saccharomycessp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha,Aspergillus niger, and mammalian cells and cell lines and insect cellsand cell lines. Thus, the invention also includes nucleic acids andpolypeptides optimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding a xylanase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the xylanase was derived, a yeast, a fungi, a plant cell, aninsect cell or a mammalian cell. Methods for optimizing codons are wellknown in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int.J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., a xylanase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides methods of making and using these transgenic non-humananimals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats and mice, comprising the nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studyxylanase activity, or, as models to screen for agents that change thexylanase activity in vivo. The coding sequences for the polypeptides tobe expressed in the transgenic non-human animals can be designed to beconstitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice whose cellsexpress proteins related to the pathology of Alzheimer's disease. U.S.Pat. No. 6,187,992, describes making and using a transgenic mouse whosegenome comprises a disruption of the gene encoding amyloid precursorprotein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing a xylanase of the invention, or, a fusion proteincomprising a xylanase of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a xylanase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides plant products, e.g., oils, seeds, leaves, extracts andthe like, comprising a nucleic acid and/or a polypeptide (e.g., axylanase) of the invention. The transgenic plant can be dicotyledonous(a dicot) or monocotyledonous (a monocot). The invention also providesmethods of making and using these transgenic plants and seeds. Thetransgenic plant or plant cell expressing a polypeptide of the presentinvention may be constructed in accordance with any method known in theart. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's xylanase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of xylanase. The can changexylanase activity in a plant. Alternatively, a xylanase of the inventioncan be used in production of a transgenic plant to produce a compoundnot naturally produced by that plant. This can lower production costs orcreate a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., axylanase) of the invention. The desired effects can be passed to futureplant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., a xylanase orantibody) of the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity) to an exemplary sequence of the invention, e.g., proteinshaving a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:134; SEQID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144;NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164,SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ IDNO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192,SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ IDNO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220,SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ IDNO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248,SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ IDNO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276,SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ IDNO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304,SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ IDNO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332,SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ IDNO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360,SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ IDNO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378 orSEQ ID NO:380. In one aspect, the polypeptide has a xylanase activity,e.g., can hydrolyze a glycosidic bond in a polysaccharide, e.g., axylan. In one aspect, the polypeptide has a xylanase activity comprisingcatalyzing hydrolysis of internal β-1,4-xylosidic linkages. In oneaspect, the xylanase activity comprises an endo-1,4-beta-xylanaseactivity. In one aspect, the xylanase activity comprises hydrolyzing axylan to produce a smaller molecular weight xylose and xylo-oligomer. Inone aspect, the xylan comprises an arabinoxylan, such as a water solublearabinoxylan.

The polypeptides of the invention include xylanases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude xylanases inactive for other reasons, e.g., before “activation”by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation or a sulfation, a dimerization event, and thelike. The polypeptides of the invention include all active forms,including active subsequences, e.g., catalytic domains or active sites,of the xylanase.

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated orrecombinant signal sequences, prepro sequences and catalytic domains(e.g., “active sites”) comprising sequences of the invention.

The percent sequence identity can be over the full length of thepolypeptide, or, the identity can be over a region of at least about 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700 or more residues. Polypeptides of the invention can also beshorter than the full length of exemplary polypeptides. In alternativeaspects, the invention provides polypeptides (peptides, fragments)ranging in size between about 5 and the full length of a polypeptide,e.g., an enzyme, such as a xylanase; exemplary sizes being of about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, ormore residues, e.g., contiguous residues of an exemplary xylanase of theinvention.

Peptides of the invention (e.g., a subsequence of an exemplarypolypeptide of the invention) can be useful as, e.g., labeling probes,antigens, toleragens, motifs, xylanase active sites (e.g., “catalyticdomains”), signal sequences and/or prepro domains.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has axylanase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, N.Y.).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′-N-C-N-R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S- form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond for mation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention includes xylanases of the invention with and withoutsignal. The polypeptide comprising a signal sequence of the inventioncan be a xylanase of the invention or another xylanase or another enzymeor other polypeptide.

The invention includes immobilized xylanases, anti-xylanase antibodiesand fragments thereof. The invention provides methods for inhibitingxylanase activity, e.g., using dominant negative mutants oranti-xylanase antibodies of the invention. The invention includesheterocomplexes, e.g., fusion proteins, heterodimers, etc., comprisingthe xylanases of the invention.

Polypeptides of the invention can have a xylanase activity under variousconditions, e.g., extremes in pH and/or temperature, oxidizing agents,and the like. The invention provides methods leading to alternativexylanase preparations with different catalytic efficiencies andstabilities, e.g., towards temperature, oxidizing agents and changingwash conditions. In one aspect, xylanase variants can be produced usingtechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce a great variety ofxylanase variants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify xylanase modulators, e.g., activators or inhibitors of xylanaseactivity. Briefly, test samples (compounds, broths, extracts, and thelike) are added to xylanase assays to determine their ability to inhibitsubstrate cleavage. Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Aswith xylanases, inhibitors can be combined to increase the spectrum ofactivity.

The enzymes of the invention are also useful as research reagents todigest proteins or in protein sequencing. For example, the xylanases maybe used to break polypeptides into smaller fragments for sequencingusing, e.g. an automated sequencer.

The invention also provides methods of discovering new xylanases usingthe nucleic acids, polypeptides and antibodies of the invention. In oneaspect, phagemid libraries are screened for expression-based discoveryof xylanases. In another aspect, lambda phage libraries are screened forexpression-based discovery of xylanases. Screening of the phage orphagemid libraries can allow the detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of phage or phagemidlibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

Another aspect of the invention is an isolated or purified polypeptidecomprising the sequence of one of Group A nucleic acid sequences andsequences substantially identical thereto, or fragments comprising atleast about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof. As discussed above, such polypeptidesmay be obtained by inserting a nucleic acid encoding the polypeptideinto a vector such that the coding sequence is operably linked to asequence capable of driving the expression of the encoded polypeptide ina suitable host cell. For example, the expression vector may comprise apromoter, a ribosome binding site for translation initiation and atranscription terminator. The vector may also include appropriatesequences for amplifying expression.

Another aspect of the invention is polypeptides or fragments thereofwhich have at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or more than about 95% homology to one of the polypeptides of Group Bamino acid sequences and sequences substantially identical thereto, or afragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,or 150 consecutive amino acids thereof. Homology may be determined usingany of the programs described above which aligns the polypeptides orfragments being compared and determines the extent of amino acididentity or similarity between them. It will be appreciated that aminoacid “homology” includes conservative amino acid substitutions such asthose described above.

The polypeptides or fragments having homology to one of the polypeptidesof Group B amino acid sequences and sequences substantially identicalthereto, or a fragment comprising at least about 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 consecutive amino acids thereof may beobtained by isolating the nucleic acids encoding them using thetechniques described above.

Alternatively, the homologous polypeptides or fragments may be obtainedthrough biochemical enrichment or purification procedures. The sequenceof potentially homologous polypeptides or fragments may be determined byxylan hydrolase digestion, gel electrophoresis and/or microsequencing.The sequence of the prospective homologous polypeptide or fragment canbe compared to one of the polypeptides of Group B amino acid sequencesand sequences substantially identical thereto, or a fragment comprisingat least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof using any of the programs describedabove.

Another aspect of the invention is an assay for identifying fragments orvariants of Group B amino acid sequences and sequences substantiallyidentical thereto, which retain the enzymatic function of thepolypeptides of Group B amino acid sequences and sequences substantiallyidentical thereto. For example the fragments or variants of saidpolypeptides, may be used to catalyze biochemical reactions, whichindicate that the fragment or variant retains the enzymatic activity ofthe polypeptides in the Group B amino acid sequences.

The assay for determining if fragments of variants retain the enzymaticactivity of the polypeptides of Group B amino acid sequences andsequences substantially identical thereto includes the steps of:contacting the polypeptide fragment or variant with a substrate moleculeunder conditions which allow the polypeptide fragment or variant tofunction and detecting either a decrease in the level of substrate or anincrease in the level of the specific reaction product of the reactionbetween the polypeptide and substrate.

The polypeptides of Group B amino acid sequences and sequencessubstantially identical thereto or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof may be used in a variety of applications. For example, thepolypeptides or fragments thereof may be used to catalyze biochemicalreactions. In accordance with one aspect of the invention, there isprovided a process for utilizing the polypeptides of Group B amino acidsequences and sequences substantially identical thereto orpolynucleotides encoding such polypeptides for hydrolyzing glycosidiclinkages. In such procedures, a substance containing a glycosidiclinkage (e.g., a starch) is contacted with one of the polypeptides ofGroup B amino acid sequences, or sequences substantially identicalthereto under conditions which facilitate the hydrolysis of theglycosidic linkage.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups and can react with many startingcompounds containing this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original smallmolecule or compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods.

In a particular aspect, the invention provides a method for modifyingsmall molecules, comprising contacting a polypeptide encoded by apolynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity isoptionally repeated. The biocatalytic reactions are conducted with agroup of biocatalysts that react with distinct structural moieties foundwithin the structure of a small molecule, each biocatalyst is specificfor one structural moiety or a group of related structural moieties; andeach biocatalyst reacts with many different small molecules whichcontain the distinct structural moiety.

Xylanase Signal Sequences, Prepro and Catalytic Domains

The invention provides xylanase signal sequences (e.g., signal peptides(SPs)), prepro domains and catalytic domains (CDs). The SPs, preprodomains and/or CDs of the invention can be isolated or recombinantpeptides or can be part of a fusion protein, e.g., as a heterologousdomain in a chimeric protein. The invention provides nucleic acidsencoding these catalytic domains (CDs), prepro domains and signalsequences (SPs, e.g., a peptide having a sequence comprising/consistingof amino terminal residues of a polypeptide of the invention). In oneaspect, the invention provides a signal sequence comprising a peptidecomprising/consisting of a sequence as set forth in residues 1 to 15, 1to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44 of a polypeptide of theinvention.

In one aspect, the invention provides a signal sequence comprising apeptide comprising/consisting of a sequence as set forth in Table 4below. For example, in reading Table 4, the invention provides a signalsequence comprising/consisting of residues 1 to 23 of SEQ ID NO:102(encoded by SEQ ID NO:101), a signal sequence comprising/consisting ofresidues 1 to 41 of SEQ ID NO:104 (encoded by SEQ ID NO:103), etc.

TABLE 4 exemplary signal sequences of the invention Signal sequence(amino acid SEQ ID NO: positions) 101, 102 1-23 103, 104 1-41 105, 1061-22 109, 110 1-26 11, 12 1-28 113, 114 1-28 119, 120 1-33 121, 122 1-20123, 124 1-20 131, 132 1-26 135, 136 1-25 139, 140 1-24 141, 142 1-25143, 144 1-32 147, 148 1-28 149, 150 1-18 15, 16 1-20 151, 152 1-21 153,154 1-16 155, 156 1-21 157, 158 1-29 159, 160 1-23 161, 162 1-32 163,164 1-26 165, 166 1-23 167, 168 1-36 169, 170 1-24 17, 18 1-31 171, 1721-29 173, 174 1-22 175, 176 1-27 177, 178 1-26 179, 180 1-19 181, 1821-25 183, 184 1-32 185, 186 1-27 187, 188 1-28 19, 20 1-29 191, 192 1-27193, 194 1-21 195, 196 1-23 197, 198 1-28 199, 200 1-30 203, 204 1-30205, 206 1-29 207, 208 1-27 209, 210 1-25 21, 22 1-28 211, 212 1-29 215,216 1-31 217, 218 1-29 219, 220 1-23 221, 222 1-24 223, 224 1-28 225,226 1-25 227, 228 1-39 229, 230 1-28 23, 24 1-29 231, 232 1-41 233, 2341-26 235, 236 1-28 237, 238 1-32 239, 240 1-30 241, 242 1-28 243, 2441-33 245, 246 1-32 249, 250 1-33 253, 254 1-24 255, 256 1-51 259, 2601-24 261, 262 1-26 263, 264 1-29 267, 268 1-30 27, 28 1-27 271, 272 1-22273, 274 1-74 277, 278 1-19 279, 280 1-22 283, 284 1-28 287, 288 1-23289, 290 1-22 295, 296 1-26 299, 300 1-24 301, 302 1-28 303, 304 1-74305, 306 1-32 309, 310 1-20 311, 312 1-33 313, 314 1-22 315, 316 1-28319, 320 1-27 325, 326 1-27 327, 328 1-29 329, 330 1-35 33, 34 1-23 331,332 1-28 333, 334 1-30 335, 336 1-50 339, 340 1-23 341, 342 1-45 347,348 1-20 349, 350 1-20 351, 352 1-73 353, 354 1-18 355, 356 1-21 357,358 1-25 359, 360 1-31 361, 362 1-26 365, 366 1-65 367, 368 1-23 369,370 1-27 39, 40 1-24 41, 42 1-37 45, 46 1-25 47, 48 1-26 5, 6 1-47 51,52 1-30 53, 54 1-37 55, 56 1-24 57, 58 1-22 59, 60 1-21 63, 64 1-20 65,66 1-22 67, 68 1-28 69, 70 1-25 7, 8 1-57 73, 74 1-21 75, 76 1-22 77, 781-27 79, 80 1-36 83, 84 1-30 87, 88 1-29 89, 90 1-40 9, 10 1-36 95, 961-24 99, 100 1-33

The xylanase signal sequences (SPs) and/or prepro sequences of theinvention can be isolated peptides, or, sequences joined to anotherxylanase or a non-xylanase polypeptide, e.g., as a fusion (chimeric)protein. In one aspect, the invention provides polypeptides comprisingxylanase signal sequences of the invention. In one aspect, polypeptidescomprising xylanase signal sequences SPs and/or prepro of the inventioncomprise sequences heterologous to a xylanase of the invention (e.g., afusion protein comprising an SP and/or prepro of the invention andsequences from another xylanase or a non-xylanase protein). In oneaspect, the invention provides xylanases of the invention withheterologous SPs and/or prepro sequences, e.g., sequences with a yeastsignal sequence. A xylanase of the invention can comprise a heterologousSP and/or prepro in a vector, e.g., a pPIC series vector (Invitrogen,Carlsbad, Calif.).

In one aspect, SPs and/or prepro sequences of the invention areidentified following identification of novel xylanase polypeptides. Thepathways by which proteins are sorted and transported to their propercellular location are often referred to as protein targeting pathways.One of the most important elements in all of these targeting systems isa short amino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The signal sequences can vary in lengthfrom 13 to 36 amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel xylanase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

It should be understood that in some aspects xylanases of the inventionmay not have SPs and/or prepro sequences, or “domains.” In one aspect,the invention provides the xylanases of the invention lacking all orpart of an SP and/or a prepro domain. In one aspect, the inventionprovides a nucleic acid sequence encoding a signal sequence (SP) and/orprepro from one xylanase operably linked to a nucleic acid sequence of adifferent xylanase or, optionally, a signal sequence (SPs) and/or preprodomain from a non-xylanase protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro domain and/or catalyticdomains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toa xylanase) with an SP, prepro domain and/or CD. The sequence to whichthe SP, prepro domain and/or CD are not naturally associated can be onthe SP's, prepro domain and/or CD's amino terminal end, carboxy terminalend, and/or on both ends of the SP and/or CD. In one aspect, theinvention provides an isolated or recombinant polypeptide comprising (orconsisting of) a polypeptide comprising a signal sequence (SP), preprodomain and/or catalytic domain (CD) of the invention with the provisothat it is not associated with any sequence to which it is naturallyassociated (e.g., a xylanase sequence). Similarly in one aspect, theinvention provides isolated or recombinant nucleic acids encoding thesepolypeptides. Thus, in one aspect, the isolated or recombinant nucleicacid of the invention comprises coding sequence for a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention and aheterologous sequence (i.e., a sequence not naturally associated withthe a signal sequence (SP), prepro domain and/or catalytic domain (CD)of the invention). The heterologous sequence can be on the 3′ terminalend, 5′ terminal end, and/or on both ends of the SP, prepro domainand/or CD coding sequence.

Hybrid (Chimeric) Xylanases and Peptide Libraries

In one aspect, the invention provides hybrid xylanases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asxylanase substrates, receptors, enzymes. The peptide libraries of theinvention can be used to identify formal binding partners of targets,such as ligands, e.g., cytokines, hormones and the like. In one aspect,the invention provides chimeric proteins comprising a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention or acombination thereof and a heterologous sequence (see above).

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of xylanases of the invention and other peptides, includingknown and random peptides. They can be fused in such a manner that thestructure of the xylanases is not significantly perturbed and thepeptide is metabolically or structurally conformationally stabilized.This allows the creation of a peptide library that is easily monitoredboth for its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of a xylanase sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed xylanase variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, asdiscussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using, e.g., assays ofxylan hydrolysis. In alternative aspects, amino acid substitutions canbe single residues; insertions can be on the order of from about 1 to 20amino acids, although considerably larger insertions can be done.Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides xylanases where the structure of the polypeptidebackbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example a alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e. xylanase activity) although variants can be selected tomodify the characteristics of the xylanases as needed.

In one aspect, xylanases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, the xylanases of the invention can be fused to a randompeptide to form a fusion polypeptide. By “fused” or “operably linked”herein is meant that the random peptide and the xylanase are linkedtogether, in such a manner as to minimize the disruption to thestability of the xylanase structure, e.g., it retains xylanase activity.The fusion polypeptide (or fusion polynucleotide encoding the fusionpolypeptide) can comprise further components as well, including multiplepeptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

Xylanases are multidomain enzymes that consist optionally of a signalpeptide, a carbohydrate binding module, a xylanase catalytic domain, alinker and/or another catalytic domain.

The invention provides a means for generating chimeric polypeptideswhich may encode biologically active hybrid polypeptides (e.g., hybridxylanases). In one aspect, the original polynucleotides encodebiologically active polypeptides. The method of the invention producesnew hybrid polypeptides by utilizing cellular processes which integratethe sequence of the original polynucleotides such that the resultinghybrid polynucleotide encodes a polypeptide demonstrating activitiesderived from the original biologically active polypeptides. For example,the original polynucleotides may encode a particular enzyme fromdifferent microorganisms. An enzyme encoded by a first polynucleotidefrom one organism or variant may, for example, function effectivelyunder a particular environmental condition, e.g. high salinity. Anenzyme encoded by a second polynucleotide from a different organism orvariant may function effectively under a different environmentalcondition, such as extremely high temperatures. A hybrid polynucleotidecontaining sequences from the first and second original polynucleotidesmay encode an enzyme which exhibits characteristics of both enzymesencoded by the original polynucleotides. Thus, the enzyme encoded by thehybrid polynucleotide may function effectively under environmentalconditions shared by each of the enzymes encoded by the first and secondpolynucleotides, e.g., high salinity and extreme temperatures.

Enzymes encoded by the polynucleotides of the invention include, but arenot limited to, hydrolases, such as xylanases. Glycosidase hydrolaseswere first classified into families in 1991, see, e.g., Henrissat (1991)Biochem. J. 280:309-316. Since then, the classifications have beencontinually updated, see, e.g., Henrissat (1993) Biochem. J.293:781-788; Henrissat (1996) Biochem. J. 316:695-696; Henrissat (2000)Plant Physiology 124:1515-1519. There are 87 identified families ofglycosidase hydrolases. In one aspect, the xylanases of the inventionmay be categorized in families 8, 10, 11, 26 and 30. In one aspect, theinvention also provides xylanase-encoding nucleic acids with a commonnovelty in that they are derived from a common family, e.g., family 5,6, 8, 10, 11, 26 or 30, as set forth in Table 5, below.

TABLE 5 SEQ ID FAMILY 9, 10 8 1, 2 8 5, 6 8 7, 8 8 99, 100 10 11, 12 10127, 128 10 27, 28 10 97, 98 10 45, 46 10 141, 142 10 107, 108 10 129,130 10 93, 94 10 63, 64 10 25, 26 10 49, 50 10 67, 68 10 85, 86 10 29,30 10 51, 52 10 35, 36 10 147, 148 10 119, 120 10 123, 124 10 249, 25010 149, 150 10 83, 84 10 43, 44 10 133, 134 10 113, 114 10 105, 106 1075, 76 10 111, 112 10 117, 118 10 115, 116 10 125, 126 10 137, 138 10135, 136 10 69, 70 10 89, 90 10 31, 32 10 13, 14 10 65, 66 10 57, 58 1077, 78 10 73, 74 10 109, 110 10 59, 60 10 71, 72 10 139, 140 10 55, 5610 15, 16 10 131, 132 10 95, 96 10 101, 102 10 39, 40 10 143, 144 10103, 104 10 17, 18 10 53, 54 10 21, 22 10 151, 152 10 23, 24 10 121, 12210 41, 42 10 47, 48 10 247, 248 10 33, 34 10 19, 20 10 87, 88 10 81, 8210 91, 92 10 61, 62 10 37, 38 10 79, 80 10 231, 232 11 157, 158 11 189,190 11 167, 168 11 207, 208 11 251, 252 11 213, 214 11 177, 178 11 187,188 11 205, 206 11 211, 212 11 197, 198 11 209, 210 11 185, 186 11 229,230 11 223, 224 11 179, 180 11 193, 194 11 173, 174 11 217, 218 11 153,154 11 219, 220 11 183, 184 11 253, 254 11 199, 200 11 255, 256 11 155,156 11 169, 170 11 195, 196 11 215, 216 11 191, 192 11 175, 176 11 161,162 11 221, 222 11 225, 226 11 163, 164 11 159, 160 11 233, 234 11 171,172 11 203, 204 11 181, 182 11 227, 228 11 165, 166 11 257, 258 26 237,238 30 241, 242 30 239, 240 30 245, 246 30 235, 236 30 313, 314 30 345,346 10 321, 322 10 323, 324 10 315, 316 10 201, 202 10 265, 266 10 145,146 10 287, 288 10 293, 294 10 351, 352 10 311, 312 10 279, 280 10 289,290 10 283, 284 10 373, 374 10 337, 338 10 371, 372 10 291, 292 10 3, 410 307, 308 10 343, 344 10 349, 350 10 329, 330 10 355, 356 10 339, 34010 295, 296 10 333, 334 10 281, 282 10 361, 362 10 347, 348 10 319, 32010 357, 358 10 365, 366 10 273, 274 10 277, 278 10 271, 272 10 285, 28610 259, 260 10 325, 326 10 331, 332 10 359, 360 10 303, 304 10 363, 36410 305, 306 10 341, 342 10 375, 376 11 377, 378 11 379, 380 11 301, 30211 309, 310 11 263, 264 11 269, 270 11 353, 354 11 299, 300 11 367, 36811 261, 262 11 369, 370 11 267, 268 11 317, 318 11 297, 298 11 327, 3285 275, 276 6

A hybrid polypeptide resulting from the method of the invention mayexhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding hydrolase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized hydrolase activities obtained from each of theoriginal enzymes, i.e. the type of bond on which the hydrolase acts andthe temperature at which the hydrolase functions. Thus, for example, thehydrolase may be screened to ascertain those chemical functionalitieswhich distinguish the hybrid hydrolase from the original hydrolases,such as: (a) amide (peptide bonds), i.e., xylanases; (b) ester bonds,i.e., esterases and lipases; (c) acetals, i.e., glycosidases and, forexample, the temperature, pH or salt concentration at which the hybridpolypeptide functions.

Sources of the original polynucleotides may be isolated from individualorganisms (“isolates”), collections of organisms that have been grown indefined media (“enrichment cultures”), or, uncultivated organisms(“environmental samples”). The use of a culture-independent approach toderive polynucleotides encoding novel bioactivities from environmentalsamples is most preferable since it allows one to access untappedresources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

Additionally, subcloning may be performed to further isolate sequencesof interest. In subcloning, a portion of DNA is amplified, digested,generally by restriction enzymes, to cut out the desired sequence, thedesired sequence is ligated into a recipient vector and is amplified. Ateach step in subcloning, the portion is examined for the activity ofinterest, in order to ensure that DNA that encodes the structuralprotein has not been excluded. The insert may be purified at any step ofthe subcloning, for example, by gel electrophoresis prior to ligationinto a vector or where cells containing the recipient vector and cellsnot containing the recipient vector are placed on selective mediacontaining, for example, an antibiotic, which will kill the cells notcontaining the recipient vector. Specific methods of subcloning cDNAinserts into vectors are well-known in the art (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989)). In another aspect, the enzymes of theinvention are subclones. Such subclones may differ from the parent cloneby, for example, length, a mutation, a tag or a label.

In one aspect, the signal sequences of the invention are identifiedfollowing identification of novel xylanase polypeptides. The pathways bywhich proteins are sorted and transported to their proper cellularlocation are often referred to as protein targeting pathways. One of themost important elements in all of these targeting systems is a shortamino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The sequences vary in length from 13 to36 amino acid residues. Various methods of recognition of signalsequences are known to those of skill in the art. In one aspect, thepeptides are identified by a method referred to as SignalP. SignalP usesa combined neural network which recognizes both signal peptides andtheir cleavage sites. See, e.g., Nielsen (1997) “Identification ofprokaryotic and eukaryotic signal peptides and prediction of theircleavage sites.” Protein Engineering, vol. 10, no. 1, p. 1-6. It shouldbe understood that some of the xylanases of the invention may or may notcontain signal sequences. It may be desirable to include a nucleic acidsequence encoding a signal sequence from one xylanase operably linked toa nucleic acid sequence of a different xylanase or, optionally, a signalsequence from a non-xylanase protein may be desired.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Eubacteria and Archaebacteria andlower eukaryotic microorganisms such as fungi, some algae and protozoa.Polynucleotides may be isolated from environmental samples in which casethe nucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms can be used. Such enzymes may function at temperaturesabove 100° C. in terrestrial hot springs and deep sea thermal vents, attemperatures below 0° C. in arctic waters, in the saturated saltenvironment of the Dead Sea, at pH values around 0 in coal deposits andgeothermal sulfur-rich springs, or at pH values greater than 11 insewage sludge. For example, several esterases and lipases cloned andexpressed from extremophilic organisms show high activity throughout awide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are preferably already in avector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or preferably, the host cell canbe a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; and plant cells. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981) and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In another aspect, it is envisioned the method of the present inventioncan be used to generate novel polynucleotides encoding biochemicalpathways from one or more operons or gene clusters or portions thereof.For example, bacteria and many eukaryotes have a coordinated mechanismfor regulating genes whose products are involved in related processes.The genes are clustered, in structures referred to as “gene clusters,”on a single chromosome and are transcribed together under the control ofa single regulatory sequence, including a single promoter whichinitiates transcription of the entire cluster. Thus, a gene cluster is agroup of adjacent genes that are either identical or related, usually asto their function. An example of a biochemical pathway encoded by geneclusters are polyketides.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Oneaspect of the invention is to use cloning vectors, referred to as“fosmids” or bacterial artificial chromosome (BAC) vectors. These arederived from E. coli f-factor which is able to stably integrate largesegments of genomic DNA. When integrated with DNA from a mixeduncultured environmental sample, this makes it possible to achieve largegenomic fragments in the form of a stable “environmental DNA library.”Another type of vector for use in the present invention is a cosmidvector. Cosmid vectors were originally designed to clone and propagatelarge segments of genomic DNA. Cloning into cosmid vectors is describedin detail in Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press (1989). Once ligated intoan appropriate vector, two or more vectors containing differentpolyketide synthase gene clusters can be introduced into a suitable hostcell. Regions of partial sequence homology shared by the gene clusterswill promote processes which result in sequence reorganization resultingin a hybrid gene cluster. The novel hybrid gene cluster can then bescreened for enhanced activities not found in the original geneclusters.

Therefore, in a one aspect, the invention relates to a method forproducing a biologically active hybrid polypeptide and screening such apolypeptide for enhanced activity by:

-   -   1) introducing at least a first polynucleotide in operable        linkage and a second polynucleotide in operable linkage, the at        least first polynucleotide and second polynucleotide sharing at        least one region of partial sequence homology, into a suitable        host cell;    -   2) growing the host cell under conditions which promote sequence        reorganization resulting in a hybrid polynucleotide in operable        linkage;    -   3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   4) screening the hybrid polypeptide under conditions which        promote identification of enhanced biological activity; and    -   5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

Screening Methodologies and “On-line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forxylanase activity (e.g., assays such as hydrolysis of casein inzymograms, the release of fluorescence from gelatin, or the release ofp-nitroanalide from various small peptide substrates), to screencompounds as potential modulators, e.g., activators or inhibitors, of axylanase activity, for antibodies that bind to a polypeptide of theinvention, for nucleic acids that hybridize to a nucleic acid of theinvention, to screen for cells expressing a polypeptide of the inventionand the like. In addition to the array formats described in detail belowfor screening samples, alternative formats can also be used to practicethe methods of the invention. Such formats include, for example, massspectrometers, chromatographs, e.g., high-throughput HPLC and otherforms of liquid chromatography, and smaller formats, such as 1536-wellplates, 384-well plates and so on. High throughput screening apparatuscan be adapted and used to practice the methods of the invention, see,e.g., U.S. Patent Application No. 20020001809.

Capillary Arrays

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. Capillary arrays, suchas the GIGAMATRI™, Diversa Corporation, San Diego, Calif.; and arraysdescribed in, e.g., U.S. Patent Application No. 20020080350 A1; WO0231203 A; WO 0244336 A, provide an alternative apparatus for holdingand screening samples. In one aspect, the capillary array includes aplurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The lumen may be cylindrical, square, hexagonal orany other geometric shape so long as the walls form a lumen forretention of a liquid or sample. The capillaries of the capillary arraycan be held together in close proximity to form a planar structure. Thecapillaries can be bound together, by being fused (e.g., where thecapillaries are made of glass), glued, bonded, or clamped side-by-side.Additionally, the capillary array can include interstitial materialdisposed between adjacent capillaries in the array, thereby forming asolid planar device containing a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a xylanase gene. One or more, or, all the transcripts of a cell canbe measured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171—R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to a xylanase of the invention. These antibodies canbe used to isolate, identify or quantify the xylanases of the inventionor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the invention or other related xylanases.The antibodies can be designed to bind to an active site of a xylanase.Thus, the invention provides methods of inhibiting xylanases using theantibodies of the invention (see discussion above regarding applicationsfor anti-xylanase compositions of the invention).

The invention provides fragments of the enzymes of the invention,including immunogenic fragments of a polypeptide of the invention. Theinvention provides compositions comprising a polypeptide or peptide ofthe invention and adjuvants or carriers and the like.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides of Group B amino acid sequences and sequencessubstantially identical thereto or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof, may also be used to generate antibodies which bind specificallyto the polypeptides or fragments. The resulting antibodies may be usedin immunoaffinity chromatography procedures to isolate or purify thepolypeptide or to determine whether the polypeptide is present in abiological sample. In such procedures, a protein preparation, such as anextract, or a biological sample is contacted with an antibody capable ofspecifically binding to one of the polypeptides of Group B amino acidsequences and sequences substantially identical thereto, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of Group B aminoacid sequences and sequences substantially identical thereto, orfragment thereof. After a wash to remove non-specifically boundproteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays and Western Blots.

Polyclonal antibodies generated against the polypeptides of Group Bamino acid sequences and sequences substantially identical thereto, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof can be obtained by directinjection of the polypeptides into an animal or by administering thepolypeptides to an animal, for example, a nonhuman. The antibody soobtained will then bind the polypeptide itself. In this manner, even asequence encoding only a fragment of the polypeptide can be used togenerate antibodies which may bind to the whole native polypeptide. Suchantibodies can then be used to isolate the polypeptide from cellsexpressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983) and theEBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies tothe polypeptides of Group B amino acid sequences and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof. Alternatively, transgenic mice may be used to express humanizedantibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides of Group B amino acidsequences and sequences substantially identical thereto, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof may be used in screening for similarpolypeptides from other organisms and samples. In such techniques,polypeptides from the organism are contacted with the antibody and thosepolypeptides which specifically bind the antibody are detected. Any ofthe procedures described above may be used to detect antibody binding.One such screening assay is described in “Methods for MeasuringCellulase Activities”, Methods in Enzymology, Vol 160, pp. 87-116.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., xylanases) and/or antibodies of theinvention. The kits also can contain instructional material teaching themethodologies and industrial uses of the invention, as described herein.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified xylanase activity, by modifying thegenetic composition of the cell. The genetic composition can be modifiedby addition to the cell of a nucleic acid of the invention, e.g., acoding sequence for an enzyme of the invention. See, e.g., WO0229032;WO0196551.

To detect the new phenotype, at least one metabolic parameter of amodified cell is monitored in the cell in a “real time” or “on-line”time frame. In one aspect, a plurality of cells, such as a cell culture,is monitored in “real time” or “on-line.” In one aspect, a plurality ofmetabolic parameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the xylanases of the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

identity of all pathway substrates, products and intermediarymetabolites

identity of all the chemical reactions interconverting the pathwaymetabolites, the stoichiometry of the pathway reactions,

identity of all the enzymes catalyzing the reactions, the enzymereaction kinetics,

the regulatory interactions between pathway components, e.g. allostericinteractions, enzyme-enzyme interactions etc,

intracellular compartmentalization of enzymes or any othersupramolecular organization of the enzymes, and,

the presence of any concentration gradients of metabolites, enzymes oreffector molecules or diffusion barriers to their movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., axylanase message) or generating new (e.g., xylanase) transcripts in acell. This increased or decreased expression can be traced by testingfor the presence of a xylanase of the invention or by xylanase activityassays. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., axylanase) or generating new polypeptides in a cell. This increased ordecreased expression can be traced by determining the amount of xylanasepresent or by xylanase activity assays. Polypeptides, peptides and aminoacids also can be detected and quantified by any method known in theart, including, e.g., nuclear magnetic resonance (NMR),spectrophotometry, radiography (protein radiolabeling), electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,various immunological methods, e.g. immunoprecipitation,immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays,gel electrophoresis (e.g., SDS-PAGE), staining with antibodies,fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Furthermore,as discussed below in detail, one or more, or, all the polypeptides of acell can be measured using a protein array.

Industrial Applications

The xylanase enzymes of the invention can be highly selective catalysts.They can catalyze reactions with exquisite stereo-, regio- andchemo-selectivities that are unparalleled in conventional syntheticchemistry. Moreover, enzymes are remarkably versatile. The xylanaseenzymes of the invention can be tailored to function in organicsolvents, operate at extreme pHs (for example, high pHs and low pHs)extreme temperatures (for example, high temperatures and lowtemperatures), extreme salinity levels (for example, high salinity andlow salinity) and catalyze reactions with compounds that arestructurally unrelated to their natural, physiological substrates.

Detergent Compositions

The invention provides detergent compositions comprising one or morepolypeptides (e.g., xylanases) of the invention, and methods of makingand using these compositions. The invention incorporates all methods ofmaking and using detergent compositions, see, e.g., U.S. Pat. Nos.6,413,928; 6,399,561; 6,365,561; 6,380,147. The detergent compositionscan be a one and two part aqueous composition, a non-aqueous liquidcomposition, a cast solid, a granular form, a particulate form, acompressed tablet, a gel and/or a paste and a slurry form. The xylanasesof the invention can also be used as a detergent additive product in asolid or a liquid form. Such additive products are intended tosupplement or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof xylanase present in the final solution ranges from about 0.001 mg to0.5 mg per gram of the detergent composition. The particular enzymechosen for use in the process and products of this invention dependsupon the conditions of final utility, including the physical productform, use pH, use temperature, and soil types to be degraded or altered.The enzyme can be chosen to provide optimum activity and stability forany given set of utility conditions. In one aspect, the xylanases of thepresent invention are active in the pH ranges of from about 4 to about12 and in the temperature range of from about 20° C. to about 95° C. Thedetergents of the invention can comprise cationic, semi-polar nonionicor zwitterionic surfactants; or, mixtures thereof.

Xylanases of the invention can be formulated into powdered and liquiddetergents having pH between 4.0 and 12.0 at levels of about 0.01 toabout 5% (preferably 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as xylanases,cellulases, lipases or endoglycosidases, endo-beta.-1,4-glucanases,beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases,laccases, amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xyloglucanases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases. Thesedetergent compositions can also include builders and stabilizers.

The addition of xylanases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe compositions of the invention as long as the enzyme is active at ortolerant of the pH and/or temperature of the intended use. In addition,the xylanases of the invention can be used in a cleaning compositionwithout detergents, again either alone or in combination with buildersand stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A xylanase of the invention maybe included as a detergent additive. The detergent composition of theinvention may, for example, be formulated as a hand or machine laundrydetergent composition comprising a polypeptide of the invention. Alaundry additive suitable for pre-treatment of stained fabrics cancomprise a polypeptide of the invention. A fabric softener compositioncan comprise a xylanase of the invention. Alternatively, a xylanase ofthe invention can be formulated as a detergent composition for use ingeneral household hard surface cleaning operations. In alternativeaspects, detergent additives and detergent compositions of the inventionmay comprise one or more other enzymes such as a xylanase, a lipase, acutinase, another xylanase, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., alactase, and/or a peroxidase (see also, above). The properties of theenzyme(s) of the invention are chosen to be compatible with the selecteddetergent (i.e. pH-optimum, compatibility with other enzymatic andnon-enzymatic ingredients, etc.) and the enzyme(s) is present ineffective amounts. In one aspect, xylanase enzymes of the invention areused to remove malodorous materials from fabrics. Various detergentcompositions and methods for making them that can be used in practicingthe invention are described in, e.g., U.S. Pat. Nos. 6,333,301;6,329,333; 6,326,341; 6,297,038; 6,309,871; 6,204,232; 6,197,070;5,856,164.

When formulated as compositions suitable for use in a laundry machinewashing method, the xylanases of the invention can comprise both asurfactant and a builder compound. They can additionally comprise one ormore detergent components, e.g., organic polymeric compounds, bleachingagents, additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. Laundry compositions of the invention can also containsoftening agents, as additional detergent components. Such compositionscontaining carbohydrase can provide fabric cleaning, stain removal,whiteness maintenance, softening, color appearance, dye transferinhibition and sanitization when formulated as laundry detergentcompositions.

The density of the laundry detergent compositions of the invention canrange from about 200 to 1500 g/liter, or, about 400 to 1200 g/liter, or,about 500 to 950 g/liter, or, 600 to 800 g/liter, of composition; thiscan be measured at about 20° C.

The “compact” form of laundry detergent compositions of the invention isbest reflected by density and, in terms of composition, by the amount ofinorganic filler salt. Inorganic filler salts are conventionalingredients of detergent compositions in powder form. In conventionaldetergent compositions, the filler salts are present in substantialamounts, typically 17% to 35% by weight of the total composition. In oneaspect of the compact compositions, the filler salt is present inamounts not exceeding 15% of the total composition, or, not exceeding10%, or, not exceeding 5% by weight of the composition. The inorganicfiller salts can be selected from the alkali and alkaline-earth-metalsalts of sulphates and chlorides, e.g., sodium sulphate.

Liquid detergent compositions of the invention can also be in a“concentrated form.” In one aspect, the liquid detergent compositionscan contain a lower amount of water, compared to conventional liquiddetergents. In alternative aspects, the water content of theconcentrated liquid detergent is less than 40%, or, less than 30%, or,less than 20% by weight of the detergent composition. Detergentcompounds of the invention can comprise formulations as described in WO97/01629.

Xylanases of the invention can be useful in formulating various cleaningcompositions. A number of known compounds are suitable surfactantsincluding nonionic, anionic, cationic, or zwitterionic detergents, canbe used, e.g., as disclosed in U.S. Pat. Nos. 4,404,128; 4,261,868;5,204,015. In addition, xylanases can be used, for example, in bar orliquid soap applications, dish care formulations, contact lens cleaningsolutions or products, peptide hydrolysis, waste treatment, textileapplications, as fusion-cleavage enzymes in protein production, and thelike. Xylanases may provide enhanced performance in a detergentcomposition as compared to another detergent xylanase, that is, theenzyme group may increase cleaning of certain enzyme sensitive stainssuch as grass or blood, as determined by usual evaluation after astandard wash cycle. Xylanases can be formulated into known powdered andliquid detergents having pH between 6.5 and 12.0 at levels of about 0.01to about 5% (for example, about 0.1% to 0.5%) by weight. These detergentcleaning compositions can also include other enzymes such as knownxylanases, xylanases, amylases, cellulases, lipases or endoglycosidases,as well as builders and stabilizers.

In one aspect, the invention provides detergent compositions havingxylanase activity (a xylanase of the invention) for use with fruit,vegetables and/or mud and clay compounds (see, for example, U.S. Pat.No. 5,786,316).

Treating Fibers and Textiles

The invention provides methods of treating fibers and fabrics using oneor more xylanases of the invention. The xylanases can be used in anyfiber- or fabric-treating method, which are well known in the art, see,e.g., U.S. Pat. Nos. 6,261,828; 6,077,316; 6,024,766; 6,021,536;6,017,751; 5,980,581; US Patent Publication No. 20020142438 A1. Forexample, xylanases of the invention can be used in fiber and/or fabricdesizing. In one aspect, the feel and appearance of a fabric is improvedby a method comprising contacting the fabric with a xylanase of theinvention in a solution. In one aspect, the fabric is treated with thesolution under pressure. For example, xylanases of the invention can beused in the removal of stains.

The xylanases of the invention can be used to treat any cellulosicmaterial, including fibers (e.g., fibers from cotton, hemp, flax orlinen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, andtoweling, made from cotton, cotton blends or natural or manmadecellulosics (e.g. originating from xylan-containing cellulose fiberssuch as from wood pulp) or blends thereof. Examples of blends are blendsof cotton or rayon/viscose with one or more companion material such aswool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).

The textile treating processes of the invention (using xylanases of theinvention) can be used in conjunction with other textile treatments,e.g., scouring and bleaching. Scouring is the removal of non-cellulosicmaterial from the cotton fiber, e.g., the cuticle (mainly consisting ofwaxes) and primary cell wall (mainly consisting of pectin, protein andxyloglucan). A proper wax removal is necessary for obtaining a highwettability. This is needed for dyeing. Removal of the primary cellwalls by the processes of the invention improves wax removal and ensuresa more even dyeing. Treating textiles with the processes of theinvention can improve whiteness in the bleaching process. The mainchemical used in scouring is sodium, hydroxide in high concentrationsand at high temperatures. Bleaching comprises oxidizing the textile.Bleaching typically involves use of hydrogen peroxide as the oxidizingagent in order to obtain either a fully bleached (white) fabric or toensure a clean shade of the dye.

The invention also provides alkaline xylanases (xylanases active underalkaline conditions). These have wide-ranging applications in textileprocessing, degumming of plant fibers (e.g., plant bast fibers),treatment of pectic wastewaters, paper-making, and coffee and teafermentations. See, e.g., Hoondal (2002) Applied Microbiology andBiotechnology 59:409-418.

Treating Foods and Food Processing

The xylanases of the invention have numerous applications in foodprocessing industry. For example, in one aspect, the xylanases of theinvention are used to improve the extraction of oil from oil-rich plantmaterial, e.g., oil-rich seeds, for example, soybean oil from soybeans,olive oil from olives, rapeseed oil from rapeseed and/or sunflower oilfrom sunflower seeds.

The xylanases of the invention can be used for separation of componentsof plant cell materials. For example, xylanases of the invention can beused in the separation of xylan-rich material (e.g., plant cells) intocomponents. In one aspect, xylanases of the invention can be used toseparate xylan-rich or oil-rich crops into valuable protein and oil andhull fractions. The separation process may be performed by use ofmethods known in the art.

The xylanases of the invention can be used in the preparation of fruitor vegetable juices, syrups, extracts and the like to increase yield.The xylanases of the invention can be used in the enzymatic treatment(e.g., hydrolysis of xylan-comprising plant materials) of various plantcell wall-derived materials or waste materials, e.g. from cereals,grains, wine or juice production, or agricultural residues such asvegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp,and the like. The xylanases of the invention can be used to modify theconsistency and appearance of processed fruit or vegetables. Thexylanases of the invention can be used to treat plant material tofacilitate processing of plant material, including foods, facilitatepurification or extraction of plant components. The xylanases of theinvention can be used to improve feed value, decrease the water bindingcapacity, improve the degradability in waste water plants and/or improvethe conversion of plant material to ensilage, and the like.

In one aspect, xylanases of the invention are used in bakingapplications, e.g., cookies and crackers, to hydrolyze arabinoxylans andcreate non-sticky doughs that are not difficult to machine and to reducebiscuit size. Use xylanases of the invention to hydrolyze arabinoxylansis used to prevent rapid rehydration of the baked product resulting inloss of crispiness and reduced shelf-life. In one aspect, xylanases ofthe invention are used as additives in dough processing. In one aspect,xylanases of the invention are used in dough conditioning, wherein inone aspect the xylanases possess high activity over a temperature rangeof about 25-35° C. and at near neutral pH (7.0-7.5). In one aspect,dough conditioning enzymes can be inactivated at the extremetemperatures of baking (>500° F.).

In one aspect, xylanases of the invention are used as additives in doughprocessing to perform optimally under dough pH and temperatureconditions. In one aspect, an enzyme of the invention is used for doughconditioning. In one aspect, a xylanase of the invention possesses highactivity over a temperature range of 25-35° C. and at near neutral pH(7.0-7.5). In one aspect, the enzyme is inactivated at the extremetemperatures of baking, for example, >500° F.

Paper or Pulp Treatment

The xylanases of the invention can be in paper or pulp treatment orpaper deinking. For example, in one aspect, the invention provides apaper treatment process using a xylanase of the invention. In oneaspect, the xylanase of the invention is applicable both in reduction ofthe need for a chemical bleaching agent, such as chlorine dioxide, andin high alkaline and high temperature environments. In one aspect, thexylanase of the invention is a thermostable alkaline endoxylanase whichcan effect a greater than 25% reduction in the chlorine dioxiderequirement of kraft pulp with a less than 0.5% pulp yield loss. In oneaspect, boundary parameters are pH 10, 65-85° C. and treatment time ofless than 60 minutes at an enzyme loading of less than 0.001 wt %. Apool of xylanases may be tested for the ability to hydrolyze dye-labeledxylan at, for example, pH 10 and 60° C. The enzymes that test positiveunder these conditions may then be evaluated at, for example pH 10 and70° C. Alternatively, enzymes may be tested at pH 8 and pH 10 at 70° C.In discovery of xylanases desirable in the pulp and paper industrylibraries from high temperature or highly alkaline environments weretargeted. Specifically, these libraries were screened for enzymesfunctioning at alkaline pH and a temperature of approximately 45° C. Inanother aspect, the xylanases of the invention are useful in the pulpand paper industry in degradation of a lignin hemicellulose linkage, inorder to release the lignin.

Animal Feeds and Food or Feed Additives

The invention provides methods for treating animal feeds and foods andfood or feed additives using xylanases of the invention, animalsincluding mammals (e.g., humans), birds, fish and the like. Theinvention provides animal feeds, foods, and additives comprisingxylanases of the invention. In one aspect, treating animal feeds, foodsand additives using xylanases of the invention can help in theavailability of nutrients, e.g., starch, protein, and the like, in theanimal feed or additive. By breaking down difficult to digest proteinsor indirectly or directly unmasking starch (or other nutrients), thexylanase makes nutrients more accessible to other endogenous orexogenous enzymes. The xylanase can also simply cause the release ofreadily digestible and easily absorbed nutrients and sugars.

When added to animal feed, xylanases of the invention improve the invivo break-down of plant cell wall material partly due to a reduction ofthe intestinal viscosity (see, e.g., Bedford et al., Proceedings of the1st Symposium on Enzymes in Animal Nutrition, 1993, pp. 73-77), wherebya better utilization of the plant nutrients by the animal is achieved.Thus, by using xylanases of the invention in feeds the growth rateand/or feed conversion ratio (i.e. the weight of ingested feed relativeto weight gain) of the animal is improved.

The animal feed additive of the invention may be a granulated enzymeproduct which may readily be-mixed with feed components. Alternatively,feed additives of the invention can form a component of a pre-mix. Thegranulated enzyme product of the invention may be coated or uncoated.The particle size of the enzyme granulates can be compatible with thatof feed and pre-mix components. This provides a safe and convenient meanof incorporating enzymes into feeds. Alternatively, the animal feedadditive of the invention may be a stabilized liquid composition. Thismay be an aqueous or oil-based slurry. See, e.g., U.S. Pat. No.6,245,546.

Xylanases of the present invention, in the modification of animal feedor a food, can process the food or feed either in vitro (by modifyingcomponents of the feed or food) or in vivo. Xylanases can be added toanimal feed or food compositions containing high amounts of xylans, e.g.feed or food containing plant material from cereals, grains and thelike. When added to the feed or food the xylanase significantly improvesthe in vivo break-down of xylan-containing material, e.g., plant cellwalls, whereby a better utilization of the plant nutrients by the animal(e.g., human) is achieved. In one aspect, the growth rate and/or feedconversion ratio (i.e. the weight of ingested feed relative to weightgain) of the animal is improved. For example a partially or indigestiblexylan-comprising protein is fully or partially degraded by a xylanase ofthe invention, e.g. in combination with another enzyme, e.g.,beta-galactosidase, to peptides and galactose and/or galactooligomers.These enzyme digestion products are more digestible by the animal. Thus,xylanases of the invention can contribute to the available energy of thefeed or food. Also, by contributing to the degradation ofxylan-comprising proteins, a xylanase of the invention can improve thedigestibility and uptake of carbohydrate and non-carbohydrate feed orfood constituents such as protein, fat and minerals.

In another aspect, xylanase of the invention can be supplied byexpressing the enzymes directly in transgenic feed crops (as, e.g.,transgenic plants, seeds and the like), such as grains, cereals, corn,soy bean, rape seed, lupin and the like. As discussed above, theinvention provides transgenic plants, plant parts and plant cellscomprising a nucleic acid sequence encoding a polypeptide of theinvention. In one aspect, the nucleic acid is expressed such that thexylanase of the invention is produced in recoverable quantities. Thexylanase can be recovered from any plant or plant part. Alternatively,the plant or plant part containing the recombinant polypeptide can beused as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

In one aspect, the invention provides methods for removingoligosaccharides from feed prior to consumption by an animal subjectusing a xylanase of the invention. In this process a feed is formedhaving an increased metabolizable energy value. In addition to xylanasesof the invention, galactosidases, cellulases and combinations thereofcan be used. In one aspect, the enzyme is added in an amount equal tobetween about 0.1% and 1% by weight of the feed material. In one aspect,the feed is a cereal, a wheat, a grain, a soybean (e.g., a groundsoybean) material. See, e.g., U.S. Pat. No. 6,399,123.

In another aspect, the invention provides methods for utilizing xylanaseas a nutritional supplement in the diets of animals by preparing anutritional supplement containing a recombinant xylanase enzymecomprising at least thirty contiguous amino acids of an amino acid ofGroup B amino acid sequences, and administering the nutritionalsupplement to an animal to increase the utilization of xylan containedin food ingested by the animal.

In yet another aspect, the invention provides an edible pelletizedenzyme delivery matrix and method of use for delivery of xylanase to ananimal, for example as a nutritional supplement. The enzyme deliverymatrix readily releases a xylanase enzyme, such as one having an aminoacid sequence of group B amino acid sequences, or at least 30 contiguousamino acids thereof, in aqueous media, such as, for example, thedigestive fluid of an animal. The invention enzyme delivery matrix isprepared from a granulate edible carrier selected from such componentsas grain germ that is spent of oil, hay, alfalfa, timothy, soy hull,sunflower seed meal, wheat midd, and the like, that readily disperse therecombinant enzyme contained therein into aqueous media. In use, theedible pelletized enzyme delivery matrix is administered to an animal todelivery of xylanase to the animal. Suitable grain-based substrates maycomprise or be derived from any suitable edible grain, such as wheat,corn, soy, sorghum, alfalfa, barley, and the like. An exemplarygrain-based substrate is a corn-based substrate. The substrate may bederived from any suitable part of the grain, but is preferably a graingerm approved for animal feed use, such as corn germ that is obtained ina wet or dry milling process. The grain germ preferably comprises spentgerm, which is grain germ from which oil has been expelled, such as bypressing or hexane or other solvent extraction. Alternatively, the graingerm is expeller extracted, that is, the oil has been removed bypressing.

The enzyme delivery matrix of the invention is in the form of discreteplural particles, pellets or granules. By “granules” is meant particlesthat are compressed or compacted, such as by a pelletizing, extrusion,or similar compacting to remove water from the matrix. Such compressionor compacting of the particles also promotes intraparticle cohesion ofthe particles. For example, the granules can be prepared by pelletizingthe grain-based substrate in a pellet mill. The pellets prepared therebyare ground or crumbled to a granule size suitable for use as an adjuvantin animal feed. Since the matrix is itself approved for use in animalfeed, it can be used as a diluent for delivery of enzymes in animalfeed.

Preferably, the enzyme delivery matrix is in the form of granules havinga granule size ranging from about 4 to about 400 mesh (USS); morepreferably, about 8 to about 80 mesh; and most preferably about 14 toabout 20 mesh. If the grain germ is spent via solvent extraction, use ofa lubricity agent such as corn oil may be necessary in the pelletizer,but such a lubricity agent ordinarily is not necessary if the germ isexpeller extracted. In other aspects of the invention, the matrix isprepared by other compacting or compressing processes such as, forexample, by extrusion of the grain-based substrate through a die andgrinding of the extrudate to a suitable granule size.

The enzyme delivery matrix may further include a polysaccharidecomponent as a cohesiveness agent to enhance the cohesiveness of thematrix granules. The cohesiveness agent is believed to provideadditional hydroxyl groups, which enhance the bonding between grainproteins within the matrix granule. It is further believed that theadditional hydroxyl groups so function by enhancing the hydrogen bondingof proteins to starch and to other proteins. The cohesiveness agent maybe present in any amount suitable to enhance the cohesiveness of thegranules of the enzyme delivery matrix. Suitable cohesiveness agentsinclude one or more of dextrins, maltodextrins, starches, such as cornstarch, flours, cellulosics, hemicellulosics, and the like. For example,the percentage of grain germ and cohesiveness agent in the matrix (notincluding the enzyme) is 78% corn germ meal and 20% by weight of cornstarch.

Because the enzyme-releasing matrix of the invention is made frombiodegradable materials, the matrix may be subject to spoilage, such asby molding. To prevent or inhibit such molding, the matrix may include amold inhibitor, such as a propionate salt, which may be present in anyamount sufficient to inhibit the molding of the enzyme-releasing matrix,thus providing a delivery matrix in a stable formulation that does notrequire refrigeration.

The xylanase enzyme contained in the invention enzyme delivery matrixand methods is preferably a thermostable xylanase, as described herein,so as to resist inactivation of the xylanase during manufacture whereelevated temperatures and/or steam may be employed to prepare thepelletized enzyme delivery matrix. During digestion of feed containingthe invention enzyme delivery matrix, aqueous digestive fluids willcause release of the active enzyme. Other types of thermostable enzymesand nutritional supplements that are thermostable can also beincorporated in the delivery matrix for release under any type ofaqueous conditions.

A coating can be applied to the invention enzyme matrix particles formany different purposes, such as to add a flavor or nutrition supplementto animal feed, to delay release of animal feed supplements and enzymesin gastric conditions, and the like. Or, the coating may be applied toachieve a functional goal, for example, whenever it is desirable to slowrelease of the enzyme from the matrix particles or to control theconditions under which the enzyme will be released. The composition ofthe coating material can be such that it is selectively broken down byan agent to which it is susceptible (such as heat, acid or base, enzymesor other chemicals). Alternatively, two or more coatings susceptible todifferent such breakdown agents may be consecutively applied to thematrix particles.

The invention is also directed towards a process for preparing anenzyme-releasing matrix. In accordance with the invention, the processcomprises providing discrete plural particles of a grain-based substratein a particle size suitable for use as an enzyme-releasing matrix,wherein the particles comprise a xylanase enzyme encoded by an aminoacid sequence of Group B amino acid sequences or at least 30 consecutiveamino acids thereof. Preferably, the process includes compacting orcompressing the particles of enzyme-releasing matrix into granules,which most preferably is accomplished by pelletizing. The mold inhibitorand cohesiveness agent, when used, can be added at any suitable time,and preferably are mixed with the grain-based substrate in the desiredproportions prior to pelletizing of the grain-based substrate. Moisturecontent in the pellet mill feed preferably is in the ranges set forthabove with respect to the moisture content in the finished product, andpreferably is about 14-15%. Preferably, moisture is added to thefeedstock in the form of an aqueous preparation of the enzyme to bringthe feedstock to this moisture content. The temperature in the pelletmill preferably is brought to about 82° C. with steam. The pellet millmay be operated under any conditions that impart sufficient work to thefeedstock to provide pellets. The pelleting process itself is acost-effective process for removing water from the enzyme-containingcomposition.

In one aspect, the pellet mill is operated with a ⅛ in. by 2 in. die at100 lb./min. pressure at 82° C. to provide pellets, which then arecrumbled in a pellet mill crumbler to provide discrete plural particleshaving a particle size capable of passing through an 8 mesh screen butbeing retained on a 20 mesh screen.

The thermostable xylanases of the invention can be used in the pelletsof the invention. They can have high optimum temperatures and high heatresistance such that an enzyme reaction at a temperature not hithertocarried out can be achieved. The gene encoding the xylanase according tothe present invention (e.g. as set forth in any of the sequences inGroup A nucleic acid sequences) can be used in preparation of xylanases(e.g. using GSSM™ as described herein) having characteristics differentfrom those of the xylanases of Group B amino acid sequences (in terms ofoptimum pH, optimum temperature, heat resistance, stability to solvents,specific activity, affinity to substrate, secretion ability, translationrate, transcription control and the like). Furthermore, a polynucleotideof Group A nucleic acid sequences may be employed for screening ofvariant xylanases prepared by the methods described herein to determinethose having a desired activity, such as improved or modifiedthermostability or thermotolerance. For example, U.S. Pat. No.5,830,732, describes a screening assay for determining thermotoleranceof a xylanase.

Waste Treatment

The xylanases of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment. For example, in oneaspect, the invention provides a solid waste digestion process usingxylanases of the invention. The methods can comprise reducing the massand volume of substantially untreated solid waste. Solid waste can betreated with an enzymatic digestive process in the presence of anenzymatic solution (including xylanases of the invention) at acontrolled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See e.g., U.S. Pat. No. 5,709,796.

Oral Care Products

The invention provides oral care product comprising xylanases of theinvention. Exemplary oral care products include toothpastes, dentalcreams, gels or tooth powders, odontics, mouth washes, pre- or postbrushing rinse formulations, chewing gums, lozenges, or candy. See,e.g., U.S. Pat. No. 6,264,925.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising xylanases of the invention. In one exemplary process,starch-containing raw materials are disintegrated and processed to forma malt. A xylanase of the invention is used at any point in thefermentation process. For example, xylanases of the invention can beused in the processing of barley malt. The major raw material of beerbrewing is barley malt. This can be a three stage process. First, thebarley grain can be steeped to increase water content, e.g., to aroundabout 40%. Second, the grain can be germinated by incubation at 15 to25° C. for 3 to 6 days when enzyme synthesis is stimulated under thecontrol of gibberellins. In one aspect, xylanases of the invention areadded at this (or any other) stage of the process. Xylanases of theinvention can be used in any beer or alcoholic beverage producingprocess, as described, e.g., in U.S. Pat. Nos. 5,762,991; 5,536,650;5,405,624; 5,021,246; 4,788,066.

In one aspect, an enzyme of the invention is used to improvefilterability and wort viscosity and to obtain a more completehydrolysis of endosperm components. Use of an enzyme of the inventionwould also increase extract yield. The process of brewing involvesgermination of the barley grain (malting) followed by the extraction andthe breakdown of the stored carbohydrates to yield simple sugars thatare used by yeast for alcoholic fermentation. Efficient breakdown of thecarbohydrate reserves present in the barley endosperm and brewingadjuncts requires the activity of several different enzymes.

In one aspect, an enzyme of the invention has activity in slightlyacidic pH (e.g., 5.5-6.0) in, e.g., the 40° C. to 70° C. temperaturerange; and, in one aspect, with inactivation at 95° C. Activity undersuch conditions would be optimal, but are not an essential requirementfor efficacy. In one aspect, an enzyme of the invention has activitybetween 40-75° C., and pH 5.5-6.0; stable at 70° for at least 50minutes, and, in one aspect, is inactivated at 96-100° C. Enzymes of theinvention can be used with other enzymes, e.g., beta-1,4-endoglucanasesand amylases.

Medical and Research Applications

Xylanases of the invention can be used as antimicrobial agents due totheir bacteriolytic properties. Xylanases of the invention can be usedto eliminating or protecting animals from salmonellae, as described ine.g., PCT Application Nos. WO0049890 and WO9903497.

Other Industrial Applications

Xylanases of the invention can be used, including Group B amino acidsequences are used in a wide variety of food, animal feed and beverageapplications. New xylanases are discovered by screening existinglibraries and DNA libraries constructed from diverse mesophilic andmoderately thermophilic locations as well as from targeted sourcesincluding digestive flora, microorganisms in animal waste, soil bacteriaand highly alkaline habitats. Biotrap and primary enrichment strategiesusing arabinoxylan substrates and/or non-soluble polysaccharidefractions of animal feed material are also useful.

Two screening formats (activity-based and sequence-based) are used inthe discovery of novel xylanases. The activity-based approach is directscreening for xylanase activity in agar plates using a substrate such asAZO-xylan (Megazyme). Alternatively a sequence-based approach may beused, which relies on bioinformatics and molecular biology to designprobes for hybridization and biopanning. See, for example, U.S. Pat.Nos. 6,054,267, 6,030,779, 6,368,798, 6,344,328. Hits from the screeningare purified, sequenced, characterized (for example, determination ofspecificity, temperature and pH optima), analyzed using bioinformatics,subcloned and expressed for basic biochemical characterization. Thesemethods may be used in screening for xylanases useful in a myriad ofapplications, including dough conditioning and as animal feed additiveenzymes.

In characterizing enzymes obtained from screening, the exemplary utilityin dough processing and baking applications may be assessed.Characterization may include, for example, measurement of substratespecificity (xylan, arabinoxylan, CMC, BBG), temperature and pHstability and specific activity. A commercial enzyme may be used as abenchmark. In one aspect, the enzymes of the invention have significantactivity at pH≧7 and 25-35° C., are inactive on insoluble xylan, arestable and active in 50-67% sucrose.

In another aspect, utility as feed additives may be assessed fromcharacterization of candidate enzymes. Characterization may include, forexample, measurement of substrate specificity (xylan, arabinoxylan, CMC,BβG), temperature and pH stability, specific activity and gastricstability. In one aspect the feed is designed for a monogastric animaland in another aspect the feed is designed for a ruminant animal. In oneaspect, the enzymes of the invention have significant activity at pH 2-4and 35-40° C., a half-life greater than 30 minutes in gastric fluid,formulation (in buffer or cells) half-life greater than 5 minutes at 85°C. and are used as a monogastric animal feed additive. In anotheraspect, the enzymes of the invention have one or more of the followingcharacteristics: significant activity at pH 6.5-7.0 and 35-40° C., ahalf-life greater than 30 minutes in rumen fluid, formulation stabilityas stable as dry powder and are used as a ruminant animal feed additive.

Enzymes are reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Moreover, unlike traditional chemical catalysts, enzymes arehighly enantio- and regio-selective. The high degree of functional groupspecificity exhibited by enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes are also capable of catalyzing many diverse reactions unrelatedto their physiological function in nature. For example, peroxidasescatalyze the oxidation of phenols by hydrogen peroxide. Peroxidases canalso catalyze hydroxylation reactions that are not related to the nativefunction of the enzyme. Other examples are xylanases which catalyze thebreakdown of polypeptides. In organic solution some xylanases can alsoacylate sugars, a function unrelated to the native function of theseenzymes.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds. Eachbiocatalyst is specific for one functional group, or several relatedfunctional groups and can react with many starting compounds containingthis functional group. The biocatalytic reactions produce a populationof derivatives from a single starting compound. These derivatives can besubjected to another round of biocatalytic reactions to produce a secondpopulation of derivative compounds. Thousands of variations of theoriginal compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so-called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods. (For further teachings on modification ofmolecules, including small molecules, see PCT/US94/09174).

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Plate Based Endoglycosidase Enzyme Discovery:Expression Screening

Titer determination of Lambda Library: Add 1.0 μL of Lambda Zap Expressamplified library stock to 600 μL E. coli MRF′ cells (OD₆₀₀=1.0). DiluteMRF′ stock with 10 mM MgSO₄. Incubate mixture at 37° C. for 15 minutes,then transfer suspension to 5-6 mL of NZY top agar at 50° C. and gentlymix. Immediately pour agar solution onto large (150 mm) NZY media plateand allow top agar to solidify completely (approximately 30 minutes).Invert the plate. Incubate the plate at 39° C. for 8-12 hours. (Thenumber of plaques is approximated. Phage titer determined to give 50,000pfu/plate. Dilute an aliquot of Library phage with SM buffer if needed.)Substrate screening: Add Lambda Zap Express (50,000 pfu) from amplifiedlibrary to 600 μL of E. coli MRF′ cells (OD₆₀₀=1.0) and incubate at 37°C. for 15 minutes. While phage/cell suspension is incubating, add 1.0 mLof desired polysaccharide dye-labeled substrate (usually 1-2% w/v) to5.0 mL NZY top agar at 50° C. and mix thoroughly. (Solution kept at 50°C. until needed.) Transfer the cell suspension to substrate/top agarsolution and gently mix. Immediately pour solution onto large (150 mm)NZY media plate. Allow top agar to solidify completely (approximately 30minutes), then invert plate. Incubate plate at 39° C. for 8-12 hours.Observe plate for clearing zones (halos) around plaques. Core plaqueswith halos out of agar and transfer to a sterile micro tube. (A largebore 200 μL pipette tip works well to remove (core) the agar plugcontaining the desired plaque.) Resuspend phage in 500 μL SM buffer. Add20 μL chloroform to inhibit any further cell growth.Isolation of pure clones: Add 5 μL of resuspended phage suspension to500 μL of E. coli MRF′ cells (OD₆₀₀=1.0). Incubate at 37° C. for 15minutes. While phage/cell suspension is incubating, add 600 μL ofdesired polysaccharide dye-labeled substrate (usually 1-2% w/v) to 3.0mL NZY top agar at 50° C. and mix thoroughly. (Solution kept at 50° C.until needed.) Transfer cell suspension to substrate/top agar solutionand gently mix. Immediately pour solution onto small (90 mm) NZY mediaplate and allow top agar to solidify completely (approximately 30minutes), then invert plate. Incubate plate at 39° C. for 8-12 hours.Plate observed for a clearing zone (halo) around a single plaque (pureclone). (If a single plaque cannot be isolated, adjust titer and replatephage suspension.) Phage are resuspended in 500 μL SM buffer and 20 μLChloroform is added to inhibit any further cell growth.Excision of pure clone: Allow pure phage suspension to incubate at roomtemperature for 2 to 3 hours or overnight at 4° C. Add 100 μL of purephage suspension to 200 μL E. coli MRF′ cells (OD₆₀₀=1.0). Add 1.0 μL ofExAssist helper phage (>1×10⁶ pfu/mL; Stratagene). Incubate suspensionat 37° C. for 15 minutes. Add 3.0 mL of 2×YT media to cell suspension.Incubate at 37° C. for 2-2.5 hours while shaking. Transfer tube to 70°C. for 20 minutes. Transfer 50-100 μL of phagemid suspension to a microtube containing 200 μL of E. Coli Exp 505 cells (OD₆₀₀=1.0). Incubatesuspension at 37° C. for 45 minutes. Plate 100 μL of cell suspension onLB_(kan 50) media (LB media with Kanamycin 50 μg/mL). Incubate plate at37° C. for 8-12 hours. Observe plate for colonies. Any colonies thatgrow contain the pure phagemid. Pick a colony and grow a small (3-10 mL)liquid culture for 8-12 hours. Culture media is liquid LB_(kan 50).Activity verification: Transfer 1.0 mL of liquid culture to a sterilemicro tube. Centrifuge at 13200 rpm (16000 g's) for 1 minute. Discardsupernatant and add 200 μL of phosphate buffer pH 6.2. Sonicate for 5 to10 seconds on ice using a micro tip. Add 200 μL of appropriatesubstrate, mix gently and incubate at 37° C. for 1.5-2 hours. A negativecontrol should also be run that contains only buffer and substrate. Add1.0 mL absolute ethanol (200 proof) to suspension and mixed. Centrifugeat 13200 rpm for 10 minutes. Observe supernatant for color. Amount ofcoloration may vary, but any tubes with more coloration than control isconsidered positive for activity. A spectrophotometer can be used forthis step if so desired or needed. (For Azo-xylan, Megazyme, read at 590nm).RFLP of pure clones from same Libraries: Transfer 1.0 mL of liquidculture to a sterile micro tube. Centrifuge at 13200 rpm (16000 g's) for1 minute. Follow QIAprep spin mini kit (Qiagen) protocol for plasmidisolation and use 40 μL holy water as the elution buffer. Transfer 10 μLplasmid DNA to a sterile micro tube. Add 1.5 μL Buffer 3 (New EnglandBiolabs), 1.5 μL 100×BSA solution (New England Biolabs) and 2.0 μL holywater. To this add 1.0 μL Not 1 and 1.0 μL Pst 1 restrictionendonucleases (New England Biolabs). Incubate for 1.5 hours at 37° C.Add 3.0 μL 6× Loading buffer (Invitrogen). Run 15 μL of digested sampleon a 1.0% agarose gel for 1-1.5 hours at 120 volts. View the gel with agel imager. Perform sequence analysis on all clones with a differentdigest pattern.

Table 6 describes various properties of exemplary enzymes of theinvention.

TABLE 6 Significant SEQ ID NO. Topt* Tstab** pHopt* activities pl M_(w)Notes 151, 152 50° C. <1 min at 65° C. 5.5-9.0 AZO-xylan 5.7 40.2 155,156 50° C. <1 min at 65° C. 5.5-8.0 AZO-xylan 8.8 62.7 169, 170 50°C. >1 min at 65° C.; <1 min 7.0 AZO-xylan 8.7 36.7 at 85° C. 195, 19650° C. >1 min at 65° C. <10 min, 5.5 AZO-xylan 8.5 36.7 <1 min 85° C.215, 216 85° C. <3 min at 85° C. 5.5-8.0 AZO-xylan 8.6 34.8 47, 48 50°C. <0.5 min at 65° C.; <1 7.0-8.0 AZO-xylan 6.2 40.3 min at 85° C. 191,192 ³85° C.  >30 sec at 85° C. 5.5 AZO-xylan 7.8 34.6 247, 248 50° C. <1min at 65° C. 8.0 AZO-xylan 9.4 43.5 7, 8 50° C. >1 min 85° C. <5 min5.5 AZO-xylan 4.5 55.3 221, 222 50-65° C. <1 min at 75° C. 5.5 AZO-xylan8.3 34.6 163, 164 65° C. <1 min at 65° C. 7.0 AZO-xylan 6.3 36.0 19, 2037° C. <5 min at 50° C. 7.0-8.0 AZO-xylan 9.2 41.5 87, 88 37-50° C. <1min at 85° C. 8.0 AZO-xylan 5.2 36.7 81, 82 50° C. <1 min at 65° C.7.0-9.0 AZO-xylan 5.3 38.8 91, 92 50° C. <1 min at 65° C. 7-8 AZO-xylan,AZO- 5.4 39.0 CMC 61, 62 37° C. <5 min at 50° C. 7.0-9.0 AZO-xylan, AZO-5.4 40 CMC 159, 160 85° C. <30 sec at 85° C. 5.5 AZO-xylan 8.3 34.5 233,234 50° C. >30 sec <1 min at 65° C.; 7.0 AZO-xylan 8.5 35.1 <1 min at85° C. 203, 204 50-65° C. >1 min at 65° C. <5 min, 5.5 AZO-xylan 9.521.7 <1 min 85° C. 181, 182 ³85° C.  >1 min at 85° C. 5.5-8.0 AZO-xylan8.8 35.5 227, 228 65° C. >1 min at 85° C. <5 min 5.5-7.0 AZO-xylan 7.825.8 45, 46 ³45° C.  ³5 min 45° C., <0.5 min >5.5  AZO-xylan 6.7 40.4*** 55° C. 231, 232 65° C. >10 min at 50° C. 5.5-7.0 AZO-xylan 8.4 31.4129, 130 65° C. <1 min at 75° C. 5.5 AZO-xylan 5.1 116 93, 94 50° C. <1min at 60° C. 8.0-9.0 AZO-xylan 5.3 39.1 189, 190 65° C. <1 min at 65°C. 5.5 AZO-xylan 9.2 20.3 **** 49, 50 70° C. <20 min 70° C. >5  AZO-xylan 5.7 38.9 85, 86 50° C. >5 min at 85° C. 5.5-7.0 AZO-xylan 6.148.4 99, 100 50° C. <1 min at 75° C. 5.5-8.0 AZO-xylan 10.8 36.6 123,124 ³85° C.  <30 sec 100° C. 5.5-7.0 AZO-xylan 6.1 44.1 249, 250 45°C. >1 min 75° C. <10 min 5.5 AZO-xylan 5.3 93 167, 168 85° C. <5 min 85°C. 5.5 AZO-xylan 9.5 21.7 207, 208 75° C. <5 min 65° C. 5.5 AZO-xylan9.1 20.4 251, 252 65-75° C. <1 min 85° C. 5.5 AZO-xylan 8.8 20.4 *****11, 12 <90° C.  <40 min 70° C. >6   AZO-xylan 6.8 43.9 177, 178 65° C.<1 min at 75° C. 5.5 AZO-xylan 8.7 44.6 9, 10 50° C. <1 min at 65° C.5.5-7.0 AZO-xylan 4.9 46.1 43, 44 37° C. unstable 5.5-7.0 AZO-xylan 4.939.1 113, 114 65-75° C. <1 min at 75° C. 5.5-8.0 AZO-xylan 5 41.2 75, 7650° C. <1 min 85° C. 7.0-9.0 AZO-xylan 4.7 39.4 111, 112 37° C. >10 min50° C. 7-8 AZO-xylan 5.6 41.0 117, 118 37° C. unstable 7-8 AZO-xylan 9.153.3 115, 116 — — — AZO-xylan 8.9 50.8 125, 126 37° C. — 8.0 AZO-xylan5.3 41.1 137, 138 50° C. <30 sec at 65° C. 5.5 AZO-xylan 5.7 38.5 69, 70³85° C.  <5 min at 85° C. 5.5-9.0 AZO-xylan 6.4 58.0 205, 206 50° C. <1min at 65° C. 5.5-8   AZO-xylan 4.3 35.1 211, 212 50° C. <1 min at 65°C. 5.5 AZO-xylan 4.4 35.4 197, 198 65° C. <1 min at 65° C. 5.5 AZO-xylan8.8 20.1 31, 32 37° C. unstable 7.0 AZO-xylan 5.1 54.4 13, 14 50° C. <1min at 65° C. 7   AZO-xylan 5.5 40.0 65, 66 50° C. <1 min at 65° C. 5.5AZO-xylan, AZO- 4.8 55.5 CMC 257, 258 37° C. unstable 5.5 AZO-xylan,AZO- 5.3 100.8 barley β-glucan, AZO-CMC 57, 58 50° C. <1 min at 65° C.7.0 AZO-xylan 4.8 56.7 185, 186 50-75° C. <1 min at 80° C. 5.5 AZO-xylan8.8 23.2 243, 244 75° C. >0.5 min @ 85° C. 5.5 AZO-xylan 8.8 44.4 77, 7850° C. <5 min at 65° C., <1 min 5.5 AZO-xylan 5.3 44.5 85° C. 229, 23037° C. ³30 min 55° C., <5 min 5.5 AZO-xylan 8.7 20.6 ****** 75° C. 109,110 65° C. >0.5 min @ 75° C. 5.5 AZO-xylan 4.9 45.2 193, 194 65° C. <1min at 75° C. 5.5 AZO-xylan 5.4 29.1 173, 174 65° C. <1 min at 80° C.7.0 AZO-xylan 7.6 51.6 59, 60 37° C. <1 min at 65° C. 7.0 AZO-xylan 6.642.5 101, 102 50° C. >0.5 min @ 65° C. 7.0 AZO-xylan 8.7 41.1 55, 56 37°C. >5 min at 50° C.; <1 min 7.0 AZO-xylan 6.5 41.8 at 85° C. 15, 16 50°C. <1 min at 65° C. 7.0 AZO-xylan 6.4 40.2 131, 132 — — — AZO-xylan 5.642.1 145, 146 65-85° C. <1 min at 85° C. 5.5 AZO-xylan 5.2 43.7 219, 220— — 5.5 AZO-xylan 6.6 34.5 253, 254 65° C. >.5 min at 85° C. 5.5-7  AZO-xylan 7.8 34.6 255, 256 65° C. >1 min 65° C. <3 min 5.5-7.0AZO-xylan 8.3 35.0 *pH or temperature optima determined by initial ratesusing AZO-AZO-xylan as a substrate **thermal stability, time that enzymeretained significant activity (approx. >50%) ***Dough conditioning****GSSM ™ parent for thermal tolerance evolution for animal feedapplications *****N35D mutation made to increase low pH activity- basedon public knowledge- mutant enzyme's relative activity at pH 4significantly increased ******Dough conditioning

Example 2 GSSM™ Screen for Thermal Tolerant Mutants

The following example describes an exemplary method for screening forthermally tolerant enzymes.

Master Plates: Prepare plates for a colony picker by labeling 96 wellplates and aliquoting 200 μL LB Amp100 into each well. (˜20 ml neededper 96 well plate). After the plates are returned from the picker,remove media from row 6 from plate A. Replace with an inoculation of SEQID NO:189. Place in a humidified 37° C. incubator overnight.Assay Plates: Pin tool cultures into a fresh 96 well plate (200 μL/wellLB Amp100). Remove plastic cover and replace with Gas Permeable Seal.Place in a humidified incubator overnight. Remove the seal and replaceplastic lid. Spin cultures down in tabletop centrifuge at 3000 rpm for10 min. Remove supernatant by inversion onto a paper towel. Aliquot 45μL Cit-Phos-KCl buffer pH 6 into each well. Replace the plastic lid withan aluminum plate seal. Use a roller to get a good seal. Resuspend cellsin a plate shaker at level 6-7 for ˜30 seconds.

Place the 96 well plate in 80° C. incubator for 20 minutes. Do notstack. Thereafter, immediately remove plates to ice water to cool for afew minutes. Remove the aluminum seal and replace with a plastic lid.Add 30 μL of 2% Azo-xylan. Mix as before on the plate shaker. Incubate37° C. in a humidified incubator overnight.

Add 200 μL ethanol to each well and pipette up and down a couple oftimes to mix. As an alternative to changing tips each time, rinse in anethanol wash and dry by expelling into a paper towel. Spin the plates at3000 rpm for 10 minutes. Remove 100 μL of supernatant to a fresh 96 wellplate. Read the OD₅₉₀.

Example 3 GSSM™ Assay for Hit Verification of Thermal Tolerant Mutants

The following example describes an exemplary method for assaying forthermally tolerant enzymes.

Pin tool or pick clones into duplicate 96 well plates (200 ul/well LBAmp 100). Remove the plastic cover and replace with a Gas PermeableSeal. Place in a humidified incubator overnight. Remove the Seal andreplace with a plastic lid. Pintool the clones to solid agar. Spincultures down in tabletop centrifuge at 3000 rpm for 10 min. Remove thesupernatant by inversion onto a paper towel. Aliquot 25 μlBPER/Lysozyme/DNase solution (see below) into each well. Resuspend cellsin a plate shaker on level 6-7 for ˜30 seconds.

Incubate the plate on ice for 15 minutes. Add 20 μL of Cit-Phos-KClbuffer pH 6 into each well. Replace the plastic lid with an aluminumplate seal. Use a roller to get a good seal. Mix on a plate shaker atlevel 6-7 for ˜30 seconds.

Place one 96 well plate in an 80° C. incubator for 20 minutes and theother at 37° C. Do not stack. Immediately remove the plates to wateryice to cool for a few minutes (use a large plastic tray if needed).Remove the aluminum seal. Add 30 μl of 2% Azo-xylan. Seal with a plasticgas permeable seal. Mix as before on the plate shaker. Incubate a set of37° C. and 80° C. plates in humidified incubator at 37° C. for 2 hoursand another set for 4 hours.

After incubation, let the plate sit for ˜5 minutes at room temperature.Add 200 μL ethanol to each well and pipette up and down a couple oftimes to mix. Instead of changing tips each time, rinse in an ethanolwash and dry by expelling into a paper towel. But, use a new set of tipsfor each clone. Spin plates at 3000 rpm 10 minutes. Remove 100 μL ofsupernatant to a fresh 96 well plate. Read OD₅₉₀.

BPER/Lysozyme/DNase solution (4.74 mL total):

4.5 mL BPR

200 μL 10 mg/mL Lysozyme (made fresh in pH 6 Cit-phos-buffer)

40 μL 5 mg/mL DNase I (made fresh in pH 6 Cit-phos buffer

Example 4 Xylanase Assay with Wheat Arabinoxylan as Substrate

The following example describes an exemplary xylanase assay that can beused, for example, to determine is an enzyme is within the scope of theinvention.

SEQ ID NOS: 11, 12, 69, 70, 77, 78, 113, 114, 149, 150, 159, 160, 163,164, 167, 168, 181, 182, 197, and 198 were subjected to an assay at pH 8(Na-phosphate buffer) and 70° C. using wheat arabinoxylan as asubstrate. The enzymes were characterized as set forth in Table 7.

TABLE 7 Protein Concen- volume of SEQ ID tration lysate added # ofprotein NOS: (mg/ml) to each vial vials Units/ml* (mg/mL) U/mg 11, 12 420.5 10 163 22.0 7.4 113, 114 37 0.6 10 66 22.0 3.0 163, 164 35 0.6 10 2522.0 1.1 197, 198 23 1.0 10 31 22.0 1.4 167, 168 10 2.2 10 228 22.0 10.477, 78 47 0.5 10 29 22.0 1.3 69, 70 18 1.3 10 36 22.0 1.7 181, 182 280.8 10 24 22.0 1.1 159, 160 25 0.9 10 43 22.0 2.0 149, 150 42 0.5 10 2422.0 1.1 *Based on addition of 1 mL of water to each sample. Units areumoles xylose released per minute based on a reducing sugar assay.

Example 5 Generation of an Exemplary Xylanase of the Invention

The following example describes the generation of an exemplary xylanaseof the invention using gene site-saturation mutagenesis (GSSM™)technology, designated the “9X” variant or mutant (the nucleic acid asset forth in SEQ ID NO:377, the polypeptide sequence as set forth in SEQID NO:378).

GSSM™ was used to create a comprehensive library of point mutations inthe exemplary SEQ ID NO:190, “wild-type” xylanase (encoded by SEQ IDNO:189). The xylanase thermotolerance screen described above identifiednine single site amino acid mutants (FIG. 6A) (D8F, Q11H, N12L, G17I,G60H, P64V, S65V, G68A & S79P) that had improved thermal tolerancerelative to the wild type enzyme (as measured following a heat challengeat 80° C. for 20 minutes). Wild-type enzyme and all nine single siteamino acid mutants were produced in E. coli and purified utilizing anN-terminal hexahistidine tag. There was no noticeable difference inactivity due to the tag.

FIG. 6 illustrates the nine single site amino acid mutants of “variant9X”, or, as set forth in SEQ ID NO:378 (encoded by SEQ ID NO:377), asgenerated by Gene Site Saturation Mutagenesis (GSSM™) of the exemplarySEQ ID NO:190 “wild-type” enzyme (encoded by SEQ ID NO:189). FIG. 6A isa schematic diagram illustrating position, numbering and the amino acidchange for the thermal tolerant point mutants of the “wild-type” gene(SEQ ID NO:190, encoded by SEQ ID NO:189). A library of all 64 codonswas generated for every amino acid position in the gene (˜13,000mutants) and screened for mutations that increased thermal tolerance.The “9X” variant was generated by combining all 9 single-site mutantsinto one enzyme. The corresponding melting temperature transitionmidpoint (T_(m)) determined by DSC for each mutant enzyme and the “9X”(SEQ ID NO:378) variant is shown on the right. FIG. 6B illustrates theunfolding of the “wild-type” (SEQ ID NO:190) and “9X” (SEQ ID NO:378)“variant/mutant” enzymes was monitored by DSC at a scan rate of 1°C./min. Baseline subtracted DSC data were normalized for proteinconcentration.

Xylanase Activity Assays

Enzymatic activities were determined using 400 ∞L of 2% Azo-xylan assubstrate in 550 ∞L of CP (citrate-phosphate) buffer, pH 6.0 at theindicated temperatures. Activity measurements as a function of pH weredetermined using 50 mM Britton and Robinson buffer solutions (pH 3.0,5.0, 6.0, 7.0, 8.0 and 9.0) prepared by mixing solutions of 0.1 Mphosphoric acid solution, 0.1 M boric acid and 0.1 M acetic acidfollowed by pH adjustment with 1 M sodium hydroxide. Reactions wereinitiated by adding 50 ∞L of 0.1 mg/ml of purified enzyme. Time pointswere taken from 0 to 15 minutes where 50 ∞L of reaction mixture wasadded to 200 ∞L of precipitation solution (100% ethanol). When all timepoints had been taken, samples were mixed, incubated for 10 minutes andcentrifuged at 3000 g for 10 minutes at 4° C. Supernatant (150 ∞L) wasaliquoted into a fresh 96 well plate and absorbance was measured at 590nm. A₅₉₀ values were plotted against time and the initial rate wasdetermined from the slope of the line.

Differential Scanning Calorimetry (DSC)

Calorimetry was performed using a Model 6100 Nano II DSC apparatus(Calorimetry Sciences Corporation, American Fork, Utah) using the DSCRunsoftware package for data acquisition, CpCalc for analysis, CpConvertfor conversion into molar heat capacity from microwatts andCpDeconvolute for deconvolution. Analysis was carried out with 1 mg/mlrecombinant protein in 20 mM potassium phosphate (pH 7.0) and 100 mM KClat a scan rate of 1° C./min. A constant pressure of 5 atm was maintainedduring all DSC experiments to prevent possible degassing of the solutionon heating. The instrumental baseline was recorded routinely before theexperiments with both cells filled with buffer. Reversibility of thethermally induced transitions was tested by reheating the solution inthe calorimeter cell immediately after cooling the first run.

Thermal Tolerance Determination

All enzymes were analyzed for thermal tolerance at 80° C. in 20 mM,potassium phosphate (pH 7.0) and 100 mM KCl. The enzymes were heated at80° C. for 0, 5, 10 or 30 minutes in thin-walled tubes and were cooledon ice. Residual activities were determined with Azo-xylan as substrateusing the assay described above for activity measurement.

Polysaccharide Fingerprinting

Polysaccharide fingerprints were determined by polysaccharide analysisusing carbohydrate gel electrophoresis (PACE). Beechwood xylan (0.1mg/mL, 100 xL, Sigma, Poole, Dorset, UK) or xylooligosaccharides (1 mM,20 ∞L, Megazyme, Wicklow, Ireland) were treated with enzyme (1-3 ∞g) ina total volume of 250 ∞L for 16 hours. The reaction was buffered in 0.1M ammonium acetate pH 5.5. Controls without substrates or enzymes wereperformed under the same conditions to identify any unspecific compoundsin the enzymes, polysaccharides/oligosaccharides or labeling reagents.The reactions were stopped by boiling for 20 min. Assays wereindependently performed at least 2 times for each condition.Derivatization using ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid,Molecular Probes, Leiden, The Netherlands), electrophoresis and imagingwere carried out as described (Goubet, F., Jackson, P., Deery, M. andDupree, P. (2002) Anal. Biochem. 300, 53-68).

Fitness Calculation

The fitness (F_(n)), for a given enzyme variant, n, was calculated byequally weighting increase in denaturation temperature transitionmidpoint (T_(m)) and increase (or decrease) in enzymatic activityrelative to the largest difference in each parameter across allvariants: F_(n)=F_(Tn)+F_(Vn), where F_(Tn)=T_(m) fitness factor of thevariant and F_(Vn)=activity fitness factor of the variant. The fitnessfactors for each (T_(m) and activity) are relative to the largestdifference in T_(m) or rate across all of the variants.F_(Tn)=(T_(m)−T_(mL))/(T_(mH)−T_(mL)) where T_(mn) is the T_(m) for thegiven variant, n, and T_(mL) is the lowest T_(m) across all variants andT_(mH) the highest T_(m) across all variants andF_(Vn)=(V_(n)−V_(L))/(V_(H)−V_(L)) where V_(n) is the relative rate forthe given variant, n, and V_(L) is the lowest rate across all variantsand V_(H) the highest rate across all variants.

Evolution by the GSSM™ Method

GSSM™ technology was used to create a comprehensive library of pointmutations in the exemplary xylanase of the invention SEQ ID NO:190(encoded by SEQ ID NO:189); including the exemplary xylanase of theinvention SEQ ID NO:378 (encoded by SEQ ID NO:377). The xylanasethermotolerance screen described above identified nine single site aminoacid mutants (FIG. 6A), D8F, Q11H, N12L, G17I, G60H, P64V, S65V, G68A &S79P, that had improved thermal tolerance relative to the exemplary“wild type” enzyme SEQ ID NO:190 (encoded by SEQ ID NO:189), as measuredfollowing a heat challenge at 80° C. for 20 minutes. Wild-type enzymeand all nine single site amino acid mutants were produced in E. coli andpurified utilizing an N-terminal hexahistidine tag. There was nonoticeable difference in activity due to the tag.

To determine the effect of the single amino acid mutations on enzymaticactivity, all nine mutants were purified and their xylanase activity(initial rates at the wild-type temperature optimum, 70° C.) wascompared to that of the exemplary SEQ ID NO:190 “wild-type” enzyme.Enzyme activities were comparable to wild type (initial rate normalizedto 1.0) for D8F, N12L, G17I, G60H, P64V, S65V G68A and S79P mutants(relative initial rates 0.65, 0.68, 0.76, 1.1, 1.0, 1.2, 0.98 and 0.84respectively) confirming that these mutations do not significantly alterthe enzymatic activity. Initial rates were measured 3 or more times andvariance was typically less than 10%. In contrast to these eightmutants, a notable reduction in enzymatic activity was observed for thebest thermal tolerant, single site mutant, Q11H (relative initial rate0.35).

Melting Temperature (T_(m)) of “Wild-Type” and Thermal Tolerant SingleSite Amino Acid Mutant Enzymes

The purified SEQ ID NO:190 “wild-type” xylanase and the nine thermaltolerant single site amino acid mutants were analyzed using differentialscanning calorimetry (DSC). Aggregation was apparent for the wild-typeenzyme as evidenced by a shoulder in the DSC trace for its thermaldenaturation, see FIG. 6B. The evolved mutant enzymes showed noindication of aggregation. For all enzymes, thermally induceddenaturation was irreversible and no discernible transition was observedin a second scan of the sample. Due to the irreversibility ofdenaturation, only the apparent T_(m) (melting temperature) could becalculated (as described, e.g., by Sanchez-Ruiz (1992) Biophys. J.61:921-935; Beldarrain (2000) Biotechnol. Appl. Biochem. 31:77-84). TheT_(m) of the wild-type enzyme was 61° C. while the T_(m)'s of all pointmutants were increased and ranged from 64° C. to 70° C. (FIG. 6A). TheQ11H mutation introduced the largest increase (T_(m)=70° C.) overwild-type followed by P64V (69° C.), G171 (67° C.) and D8F (67° C.).

The “9X” combined GSSM™ Exemplary Enzyme SEQ ID NO:378

The “9X” enzyme (SEQ ID NO:378) was constructed by combining thesingle-site changes of the nine thermal tolerant up-mutants bysite-directed mutagenesis (FIG. 6A). The “9X” (SEQ ID NO:378) enzyme wasexpressed in E. coli and purified to homogeneity. DSC was performed todetermine the melting temperature. The T_(m) of “9X” enzyme was 34degrees higher than SEQ ID NO:190, the “wild-type” enzyme, demonstratinga dramatic shift in its thermal stability (FIG. 6B).

To evaluate the effect of the combined mutations and elevated meltingtemperature on the enzyme's biochemical properties, pH and temperatureprofiles were constructed and compared to SEQ ID NO:190, the “wild-type”enzyme. FIG. 7 illustrates the biochemical characterization of “wildtype” and “evolved” 9X mutant enzymes. FIG. 7A illustrates thepH-dependence of activity for the wild-type and evolved 9X mutantenzymes. Xylanase activity was measured at 37° C. at each pH and theinitial velocity was plotted against absorbance at 590 nm to determineinitial rates. FIG. 7B illustrates the temperature-dependence ofactivity for the wild-type and evolved 9X mutant enzymes. The optimumtemperatures of the wild-type and 9X mutant enzymes were measured over atemperature range of 25-100° C. at pH 6.0 and are based on initial ratesmeasured over 5 minutes. FIG. 7C illustrates the thermal stability ofwild-type and evolved 9X mutant enzymes. Thermal dependence of activityof the wild-type and evolved 9X mutant enzymes was measured by firstheating the enzymes at each of the indicated temperatures for 5 minutesfollowed by cooling to room temperature and the measurement of residualactivity (initial rate at 37° C., pH 6.0). For all experiments initialrates were measured 2 or more times and the variation was less than 10%.

SEQ ID NO:190 and SEQ ID NO:378 (the “9X” mutant) enzyme had comparablepH/activity profiles with the highest activity between pH 5 and 6 (FIG.7A). Both enzymes had similar initial rate/temperature optima at 70° C.,however, SEQ ID NO:190, the “wild-type” enzyme had higher activity atlower temperatures (25-50° C.) whereas SEQ ID NO:378 (the “9X” mutant)retained more than 60% of its activity up to 100° C. (determined byinitial rate) in the presence of substrate (FIG. 7B). The activity ofSEQ ID NO:190, the “wild-type” enzyme was not detectable at temperaturesabove 70° C.

To determine the effect of the 9 combined mutations on enzyme thermaltolerance, residual activity was measured and compared to SEQ ID NO:190,the “wild-type” enzyme. Residual activity was determined by a heatchallenge for 5 minutes at each temperature (37, 50, 60, 70, 80 and 90°C.) followed by activity measurements at 37° C. SEQ ID NO:190 wascompletely inactivated above 70° C. while the evolved 9X mutantdisplayed significant activity after heating at 70, 80 and even 90° C.(FIG. 7C). Furthermore, although the activity of the wild-type enzymedecreased with increasing temperature, the 9X variant was somewhatactivated by heating at temperatures up to 60° C.

Generation of Combinatorial GSSM™ Variants Using GeneReassembly™Technology

To identify combinatorial variants of the 9 single site amino acidmutants with highest thermal tolerance and activity compared to theadditively constructed SEQ ID NO:378 (the “9X” variant), aGeneReassembly™ library (U.S. Pat. No. 6,537,776) of all possible mutantcombinations (2⁹) was constructed and screened. Using thermal toleranceas the screening criterion, 33 unique combinations of the nine mutationswere identified as was the original 9X variant. A secondary screen wasperformed to select for variants with higher activity/expression thanthe evolved 9X. This screen yielded 10 variants with sequencespossessing between 6 and 8 of the original single mutations in variouscombinations, as illustrated in FIG. 8A. FIG. 8 illustrates thecombinatorial variants identified using GeneReassembly™ technology. FIG.8A illustrates the GeneReassembly™ library of all possible combinationsof the 9 GSSM™ point mutations that was constructed and screened forvariants with improved thermal tolerance and activity. Eleven variantsincluding the 9X variant were obtained. As shown in the figure, thevariants possessed 6, 7, 8, or 9 of the point mutations in variouscombinations. The corresponding melting temperature transition midpoint(Tm) determined by DSC of each variant is shown on the right. FIG. 8Billustrates the relative activity (initial rate measured over a 5 minutetime period) of the 6X-2 and 9X variants compared to wild-type at thetemperature optimum (70° C.) and pH 6.0. Error bars show the range inthe initial rate for 3 measurements.

The melting temperature (T_(m)) of each of the combinatorial variantswas at least 28° C. higher than wild type (FIG. 8A) and all of thereassembly variants displayed higher relative activity than the 9Xenzyme. The activity of one variant in particular, 6X-2, was greaterthan the wild-type enzyme and significantly better (1.7X) than the 9Xenzyme (FIG. 8B). Sequence comparison of the reassembly variantsidentified at least 6 mutations that were required for the enhancedthermostability (>20 degrees). All 33 unique variants found in theinitial thermostability screen contained both Q11H and G171 mutationsdemonstrating their importance for thermal tolerance.

Analysis of Wild-Type and Variant Polysaccharide Product Fingerprints

The products generated by the “wild-type,” 6X-2 and 9X variants werecompared by polysaccharide analysis using carbohydrate gelelectrophoresis (PACE). Different substrates (oligosaccharides andpolysaccharides) were tested for hydrolysis by the xylanases. Thedigestion products of the 3 xylanases tested were very similar, asillustrated in FIG. 9. All three enzymes hydrolyzed (Xyl)₆ and (Xyl)₅,mainly into both (Xyl)₃ and (Xyl)₂, and (Xyl)₄ was hydrolyzed to (Xyl)₂(FIG. 9A). Only a small amount of hydrolysis of (Xyl)₃ into (Xyl)₂ andXyl was observed indicating that (Xyl)₃ is a relatively poor substratefor the enzyme. No activity was detected on (Xyl)₂. Beechwood xylan,which contains glucuronosyl residues, was hydrolyzed by all threeenzymes mainly into (Xyl)₂ and (Xyl)₃, but other bands were detectedthat migrated between oligoxylan bands (FIG. 9B). In PACE analysis, eacholigosaccharide has a specific migration depending on the sugarcomposition and degree of polymerization (Goubet, F., Jackson, P.,Deery, M. and Dupree, P. (2002) Anal. Biochem. 300, 53-68), thus, thesebands likely correspond to oligoglucuronoxylans. Therefore, the evolvedenzymes retained the substrate specificity of the “wild-type” enzyme.

As noted above, FIG. 9 illustrates the product fingerprints of“wild-type” SEQ ID NO:190 (encoded by SEQ ID NO:189), 6X-2 (SEQ IDNO:380, encoded by SEQ ID NO:379) and SEQ ID NO:378 (the “9X” mutant)enzyme variant, as determined by PACE. FIG. 9A illustrates fingerprintsobtained after hydrolysis of oligoxylans (Xyl)₃, (Xyl)₄, (Xyl)₅ and(Xyl)₆ by “wild-type” and variant enzymes. Control lanes containoligosaccharide incubated under the assay conditions in the absence ofenzyme. FIG. 9B illustrates the fingerprints obtained after hydrolysisof Beechwood xylan by wild-type and variant enzymes. Standards contained(Xyl)₂, (Xyl)₃, (Xyl)₄. All assays were performed at 37° C. and pH 5.5.

A combination of laboratory gene evolution strategies was used torapidly generate a highly active, thermostable xylanase optimized forprocess compatibility in a number of industrial market applications.GSSM™ methodology was employed to scan the entire sequence of theexemplary “wild type” xylanase SEQ ID NO:190 (encoded by SEQ ID NO:189)and to identify 9 point mutations that improve its thermal tolerance.Although it had no discernable effect on the hydrolysis product profileof the enzyme, as illustrated in FIG. 9, the addition of the 9 mutationsto the protein sequence resulted in a moderate reduction in enzymaticspecific activity at SEQ ID NO:190 (the “wild-type”)'s temperatureoptimum. 70° C., see FIG. 9B. Using the GeneReassembly™ method togenerate a combinatorial library of the 9 single site amino acidmutants, this reduction in activity was overcome. Ten thermostablevariants (T_(m)'s between 89° C. and 94° C.) with activity better thanthe “9X” variant were obtained from screening the GeneReassembly™library. With a T_(m) of 90° C., enzymatic specific activity surpassingwild-type and a product fingerprint unaltered and comparable to SEQ IDNO:190 (the “wild-type”), the 6X-2 variant (SEQ ID NO:380, encoded bySEQ ID NO:379) is particularly notable. To our knowledge the shift inT_(m) obtained for these variants is the highest increase reported fromthe application of directed evolution technologies.

SEQ ID NO:380 (the 6X-2 variant) includes the following changes, ascompared to SEQ ID NO:190 (the “wild-type”): D8F, Q11H, G17I, G60H, S65Vand G68A. SEQ ID NO:379 includes the following nucleotide changes, ascompared to the “wild type” SEQ ID NO:189: the nucleotides at positions22 to 24 are TTC, the nucleotides at positions 31 to 33 are CAC, thenucleotides at positions 49 to 51 are ATA, the nucleotides at positions178 to 180 are CAC, the nucleotides at positions 193 to 195 are GTG, thenucleotides at positions 202 to 204 are GCT.

In order to gauge the effectiveness of combinatorial mixing versusaddition of the point mutants to the desired phenotype, a fitnessparameter combining contributions both from changes in enzyme activityand thermostability was calculated for each mutant. The term fitness asdescribed here is not an objective measure that can be compared to otherenzymes, but rather a term that allows the measurement of the success ofdirected evolution of this particular xylanase. Since enzyme fitness, F,is calculated by equally weighting changes in T_(m) and enzyme activityfor this set of variants, the maximum allowable fitness value is 2(F_(T)≦1 and F_(V)≦1, see above). In other words, if the variant withthe best activity also had the highest T_(m), its fitness value would be2. With a fitness value near 2 (see FIG. 10B), the 6X-2 variant (SEQ IDNO:380, encoded by SEQ ID NO:379) is the closest to possessing the bestpossible combination of thermal stability and enzyme activity. Thesingle site mutation that confers the highest value of fitness is S65V.Although the T_(m) of the S65V mutant is lower than that of the Q11Hmutant (66° C. verses 70° C. respectively), it has a higher fitnessvalue since its specific activity is not reduced relative to wild-type.

FIG. 10A is a schematic diagram illustrating the level of thermalstability (represented by T_(m)) improvement over “wild-type” obtainedby GSSM™ evolution. The single site amino acid mutant and thecombinatorial variant with the highest thermal stability (Q11H and “9X”(SEQ ID NO:378), respectively) are shown in comparison to wild-type.FIG. 10B illustrates a “fitness diagram” of enzyme improvement obtainedby combining GSSM™ and GeneReassembly™ technologies. Fitness wasdetermined using the formula F=FT+FV where fitness (F) is calculated byequally weighting thermal tolerance fitness (FT) and relative activityfitness (FV) as described above. The point mutation that confers thegreatest fitness (S65V) is shown. Combining all 9 point mutations alsoimproved fitness (SEQ ID NO:378, the “9X” variant). However, the largestimprovement in fitness was obtained by combining GSSM™ andGeneReassembly™ methods to obtain the best variant, 6X-2 (SEQ IDNO:380).

The GeneReassembly™ method also allowed the identification of importantresidues that appear absolutely necessary for improved thermalstability. Two key residues, Q11H and G17I, were present in everyGeneReassembly™ variant identified based on thermal tolerance (see FIG.6A). The structural determinants for thermal stability of proteins havebeen studied and several theories have been documented, e.g., by Kinjo(2001) Eur. Biophys. J. 30:378-384; Britton (1999) J. Mol. Biol.293:1121-1132; Ladenstein (1998) Adv. Biochem. Eng. Biotechnol.61:37-85; Britton (1995) Eur. J. Biochem. 229:688 695; Tanner (1996)Biochemistry 35:2597-2609; Vetriani (1998) Proc. Natl. Acad. Sci. USA95:2300-2305. Hydrogen bonding patterns, ionic interactions, hydrophobicpacking and decreased length of surface loops are among the key factorseven though the contribution of each to protein stability is not fullyunderstood. Given that most of the beneficial point substitutionsidentified from testing all possible single amino acid substitutionsinvolved the replacement of relatively polar, charged or small (glycine)residues for much larger hydrophobic residues, it can surmised thathydrophobic interactions play the most significant role in enhancing thethermostability of this protein. Even with a good understanding of theoptimal interactions to enhance thermal tolerance, the prediction ofwhere to make mutations that introduce such interactions is notstraightforward. A nonrational approach using the GSSM™ method, however,allows rapid sampling of all sidechains at all positions within aprotein structure. Such an approach leads to the discovery of amino acidsubstitutions that introduce functional interactions that could not havebeen foreseen.

Example 6 Pre-Treating Paper Pulp with Xylanases of the Invention

In one aspect, xylanases of the invention can be used to pretreat paperpulp. This example describes an exemplary routine screening protocol todetermine whether a xylanase is useful in pretreating paper pulp; e.g.,in reducing the use of bleaching chemicals (e.g., chlorine dioxide,ClO₂) when used to pretreat Kraft paper pulp.

The screening protocol has two alternative test parameters: Impact ofxylanase treatment after an oxygen delignification step (Post-O₂ pulp);and, Impact of xylanase in a process that does not include oxygendelignification (pre-O₂ brownstock).

For pulp treatment conditions that simulate process conditions inindustrial situations, e.g., factories: pH 8.0; 70° C.; 60 min duration.

The process is schematically depicted in the Flow Diagram of FIG. 11.

Twenty xylanases were identified by biochemical tests that were activeunder these conditions. Of the 20 xylanases, 6 were able tosignificantly reduce ClO₂ demand when they were used to pretreat Kraftpulp before it was chemically bleached. The six are: SEQ ID NO:182(encoded by SEQ ID NO:181); SEQ ID NO:160 (encoded by SEQ ID NO:159);SEQ ID NO:198 (encoded by SEQ ID NO:197); SEQ ID NO:168 (encoded by SEQID NO:167); SEQ ID NO:216 (encoded by SEQ ID NO:215); SEQ ID NO:260(encoded by SEQ ID NO:259). Others showed some activity but were not asgood. Xylanases SEQ ID NO:182 (encoded by SEQ ID NO:181) and SEQ IDNO:160 (encoded by SEQ ID NO:159) are modular and contain a carbohydratebinding module in addition to the xylanase catalytic domain. It wasdemonstrated that truncated derivatives of these 2 xylanases containingjust the catalytic domain are more effective in this application. Thebest xylanase, SEQ ID NO:160 (encoded by SEQ ID NO:159) was studied morecomprehensively. Results can be summarized as follows:

-   -   pretreatment of post-O₂ spruce/pine/fir (SPF) pulp with 2        units/g of SEQ ID NO:160 (encoded by SEQ ID NO:159) reduces        subsequent ClO₂ use by 22% to reach 65% GE brightness;    -   pretreatment of pre-O₂ brownstock SPF with 0.5 units/g SEQ ID        NO:160 (encoded by SEQ ID NO:159) reduces subsequent ClO₂ use by        13% to reach 65% GE brightness;    -   pretreatment of pre-O₂ Aspen pulp with 0.5 units/g SEQ ID NO:160        (encoded by SEQ ID NO:159) reduces ClO₂ use by at least 22%;    -   pretreatment of pre-O₂ Douglas Fir/Hemlock pulp with 0.5 units/g        SEQ ID NO:160 (encoded by SEQ ID NO:159) reduces ClO₂ use by at        least 22%;    -   under the treatment conditions employed, the reduction in yield        from the xylanase treatment did not exceed 0.5% when compared        with pulp that had been bleached at the same kappa factor but        not treated with xylanase;    -   optimal conditions for treating post-O₂SPF pulp with SEQ ID        NOS:159, 160 were: pH 6-7, enzyme dose 0.3 units/g, treatment        time 20-25 min. Under these conditions, reduction in ClO₂ use of        28% was possible to reach 69% GE brightness.

In further experiments:

SEQ ID NO:160 (XYLA), encoded by SEQ ID NO:159=full length wild typexylanase:

-   -   XYLA (E.c)=truncated variant of SEQ ID NOS:159, 160 containing        only xylanase catalytic domain expressed in E. coli    -   XYLA (P.f)=ditto but expressed in P. fluorescens

SEQ ID NO:182 (encoded by SEQ ID NO:181)-=second full-length wild typexylanase:

-   -   XYLB (E.c)=truncated variant etc, etc expressed in E. coli    -   XYLB (P.f)=ditto but expressed in P. fluorescens        Dose Response Data for Lead Xylanases on Pre-O2 Brownstock

Conditions for xylanase stage (X-stage) as follows:

pH 8

Temperature 70° C.

Time 60 min

Kappa factor 0.24

For no-enzyme control, kappa factor was 0.30

Results showed a dose dependent increase in brightness forxylanase-treated samples at a lower charge of chlorine dioxide (ClO₂)(Kf 0.24 vs Kf 0.30).

In each case, the truncated derivative looked to be more effective thatthe full-length xylanase. Optimal xylanase dose looked to be around 0.6to 0.7 U/g pulp.

Pretreatment of Intercontinental Pre-O₂ Brownstock with the Best 4Xylanases

Determination of ClO₂ Dose Response in D_(o)

Experimental outline

Pre-O₂ Brownstock

-   -   Initial kappa 31.5

X stage conditions

-   -   Xylanase charge 0.7 U/gm    -   Temperature 70° C.    -   pH8    -   Treatment time 1 hr    -   Pulp consistency 10%

Bleach sequence XDEP

-   -   Kappa factor 0.22, 0.26 and 0.30 (% D on pulp: 2.63, 3.12 and        3.60)        Final Brightness after 3-Stage Bleach Sequence Versus Kappa        Factor (ClO₂ Charge):    -   XYLB—At 61.5 final brightness, X-stage enables reduction in ClO₂        use of 3.89 kg/ton pulp.    -   XYLB (E.c)—At 61.5 final brightness, X-stage enables reduction        in ClO₂ charge of 4.07 kg/ton pulp.    -   XYLA—At 61.5 brightness, X-stage enables a reduction in ClO₂ use        of 4.07 kg/ton pulp.    -   XYLA (E.c)—At 61.5 final brightness, X-stage enables reduction        in ClO₂ use of 4.90 kg/ton pulp.        Determination of ClO₂ Dose Response in D_(o):

ClO₂ Savings in D_(o) Kf reduction in Enzyme (kg/ton OD) D_(o) XYLB 3.8911.7% XYLB (E.c) 5.08 15.8% XYLA 4.07 12.2% XYLA (E.c) 4.90 14.7%

Xylanase 0.7 U/g, pH 8.0, 70° C., 1 hr

Pulp: Pre-02 Brownstock, initial kappa 31.5

Percentage saving of ClO₂ is of little significance to the industry.Their primary concern is lbs of ClO₂ required per ton OD pulp. Thismakes sense when one considers that a lower percentage saving seen witha high initial kappa brownstock can be more valuable in terms of lbs ofClO₂ saved than a higher percentage reduction for a low initial kappapulp which will require a lower total charge of ClO₂ to reach targetbrightness.

Relationship between Brightness, Yield and Kappa Factor for BleachedControl Pulp:

The results showed that bleaching with increasing doses of ClO₂ toachieve higher target brightness results in increased loss of pulpyield. This is an issue because pulp at this stage of the process has avalue of almost $400 per ton and loss of cellulose costs money.

A benefit of xylanase (e.g., a xylanase of the invention) is that use ofa lower ClO₂ dose can reduce yield losses as long as the action of thexylanase itself doesn't cancel out the gain.

Dose Response Data for Pretreatment of Pre-O₂ Brownstock with XylanaseXYLB (P.f):

Experimental outline

Northwood Pre-O₂ Brownstock

Initial kappa 28.0

Initial consistency 32.46%

Initial brightness 28.37

X stage conditions

Xylanase charge 0 to 2.70 U/gm

Temperature 58° C. to 61° C.

pH 8.2 to 8.5

Treatment time 1 hr

Bleach sequence XDEp

Kappa factor 0.24

ClO₂ saving calculated for Kappa factors between 0.24 and 0.30

The purpose of this experiment was to evaluate the best of the 4xylanases on unwashed SPF brownstock. Results showed dose-dependentincreases in final brightness for pulp treated with XYLB (E.c), withbrightness achieved in presence of xylanase at lower Kf of 0.24,approaching brightness achieved at higher Kf of 0.30 asymptotically.

Relationship between Dose of Xylanase XYLB (E.c) and Chlorine DioxideSaving (Pre-O2 Brownstock):

ClO₂ Saving in % OD ClO₂Saving in kg/ton Pulp Pulp Xylanase Dose in U/gm0.299% 2.99 0.31 0.363% 3.63 0.51 0.406% 4.06 0.71 0.439% 4.39 0.910.483% 4.83 1.26 0.523% 5.32 1.80 0.587% 5.87 2.70

Optimum Xylanase Dose is between 0.5 and 0.9 U/gm

The optimum dose lies in the range 0.5 to 0.9 U/g. Above this dose thereis a diminishing return per unit increment of xylanase. Reductions inchlorine dioxide dose per ton of pulp treated of this magnitude arecommercially significant.

Three-Stage Biobleaching Procedure

A three-stage biobleaching procedure was developed that would closelysimulate the actual bleaching operations in a pulp mill bleach plant(FIG. 1). This bleach sequence is designated by (X) DoEp, in which Xrepresents the xylanase treatment stage, D for chlorine dioxidebleaching stage, and Ep for alkaline peroxide extraction stage. Theprimary feedstock used in our application tests was Southern SoftwoodKraft Brownstock (without oxygen delignification). The most effectivexylanase candidates that showed high bleach chemical reduction potentialin the biobleaching assays were also tested on two species of hardwoodKraft pulp (maple and aspen). Upon completion of each biobleachinground, the ensuing pulp was used to produce TAPPI (Technical Associationof Pulp and Paper Industries)-standard handsheets. The GE % brightnessof each handsheet was measured, and the brightness values were used asthe indication of how well each enzyme had performed on the pulp duringthe enzymatic pretreatment stage (X).

Results:

Out of approximately 110 xylanases that were screened using the (X) DoEpbiobleaching sequence, 4 enzymes, i.e., XYLA (P.f); XYLB (P.f); SEQ IDNO216 (encoded by SEQ ID NO:215); SEQ ID NO:176 (encoded by SEQ IDNO:175); showed the greatest potential for reducing the use of bleachingchemicals. While XYLA (P.f) and XYLB (P.f) exhibited equally highperformance (best among the four good performers), XYLA (P.f) showed abetter pH tolerance than XYLB (P.f). The results can be summarized asfollows:

-   -   It is possible to achieve a handsheet brightness of 60 (GE %)        using a three-stage bleach sequence [(X) DoEp] that involves        pretreatment of Southern Softwood Kraft Brownstock with the        following four enzymes at the loading levels listed below (pH=8,        65° C. & 1 h):        -   XYLA (P.f) at 0.55 U/g pulp        -   XYLB (P.f) at 0.75 U/g pulp        -   SEQ ID NOS:215, 216 at 1.80 U/g pulp        -   SEQ ID NOS:175, 176 at 1.98 U/g pulp    -   Pretreatment of Southern Softwood Kraft Brownstock with 2 U/g        pulp of XYLA (P.f) reduces ClO₂ use by 18.7% to reach a final GE        % brightness of 61.    -   XYLA (P.f) exhibits good tolerance at higher pH and provides        more than 14% chemical savings when the enzymatic pretreatment        stage is run at pH=10.    -   Pretreatment of Southern Softwood Kraft Brownstock with 2 U/g        pulp of XYLB (P.f) reduces ClO₂ use by 16.3% to reach a final GE        % brightness of 60.5.    -   Pretreatment of aspen Kraft pulp with 2 U/g pulp of XYLA (P.f)        and XYLB (P.f) reduces ClO₂ use by about 35% to reach a final GE        % brightness of 77.    -   Pretreatment of maple Kraft pulp with 2 U/g pulp of XYLA (P.f)        and XYLB (P.f) reduces ClO₂ use by about 38% to reach a final GE        % brightness of 79.    -   The two best performing xylanases, namely XYLA (P.f) and XYLB        (P.f), are truncated enzymes, containing just the catalytic        domain, and were produced in Pseudomonas fluorescens.

While the invention has been described in detail with reference tocertain preferred aspects thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. An isolated, synthetic or recombinant polypeptide having a xylanase activity comprising: (i) an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:160, or, (ii) an amino acid sequence encoded by a nucleic acid having at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:159; or (iii) enzymatically active polypeptide fragments of (i) or (ii).
 2. The isolated, synthetic or recombinant polypeptide of claim 1, wherein the xylanase activity comprises catalyzing hydrolysis of internal β-1,4-xylosidic linkages, comprises an endo-1,4-beta-xylanase activity, comprises hydrolyzing a xylan to produce a smaller molecular weight xylose and xylo-oligomer, comprises hydrolyzing polysaccharides comprising 1,4-β-glycoside-linked D-xylopyranoses, comprises hydrolyzing hemicelluloses, comprises hydrolyzing hemicelluloses in a wood, wood product, wood pulp, paper, paper pulp, paper product, Kraft pulp, or a combination thereof, comprises catalyzing hydrolysis of xylans in a feed or a food product, or comprises catalyzing hydrolysis of xylans in a microbial cell or a plant cell.
 3. An isolated, synthetic or recombinant polypeptide comprising the polypeptide of claim 1 and lacking a signal sequence or a prepro sequence.
 4. An isolated, synthetic or recombinant polypeptide comprising the polypeptide of claim 1 and having a heterologous sequence, a heterologous signal sequence or a heterologous prepro sequence.
 5. A protein preparation comprising the polypeptide of claim 1, wherein the protein preparation comprises a liquid, a solid or a gel.
 6. A heterodimer comprising the polypeptide of claim 1 as a first domain and a second domain, wherein the heterodimer is a fusion protein comprising the amino acid sequence of claim 1 as a first domain and additional amino acid sequence for a second domain.
 7. A homodimer comprising the polypeptide of claim 1, wherein the homodimer is a fusion protein comprising two of the amino acid sequence of claim
 1. 8. An immobilized polypeptide, wherein the polypeptide comprises the sequence of claim 1, or the heterodimer of claim
 6. 9. An array comprising the immobilized polypeptide of claim 1, or the heterodimer of claim
 6. 10. The isolated, synthetic or recombinant polypeptide of claim 1, wherein the polypeptide comprises (a) at least one glycosylation site, or (b) at least one N-linked glycosylation site.
 11. An immobilized polypeptide, wherein the polypeptide comprises the amino acid sequence of the polypeptide of claim 1, and the polypeptide is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
 12. The isolated, synthetic or recombinant polypeptide of claim 1, wherein the sequence identity to the amino acid sequence of SEQ ID NO:160 is at least 95%.
 13. The isolated, synthetic or recombinant polypeptide of claim 12, wherein the sequence identity to the amino acid sequence of SEQ ID NO:160 is at least 97%.
 14. The isolated, synthetic or recombinant polypeptide of claim 13, wherein the sequence identity to the amino acid sequence of SEQ ID NO:160 is at least 98%.
 15. The isolated, synthetic or recombinant polypeptide of claim 14, wherein the sequence identity to the amino acid sequence of SEQ ID NO:160 is at least 99%.
 16. The isolated, synthetic or recombinant polypeptide of claim 15, wherein the polypeptide has the amino acid sequence of SEQ ID NO:160.
 17. An isolated, synthetic or recombinant polypeptide comprising the amino acid sequence of the polypeptide of claim 1 and lacking a carbohydrate binding module.
 18. The isolated, synthetic or recombinant polypeptide of claim 17, also lacking a signal sequence or a prepro sequence.
 19. An isolated, synthetic or recombinant polypeptide of claim 1, lacking a homologous carbohydrate binding module, signal sequence or prepro sequence and further comprising a heterologous carbohydrate binding module, signal sequence or prepro sequence.
 20. An isolated, synthetic or recombinant polypeptide comprising the polypeptide of claim 1 and having a heterologous signal sequence, a carbohydrate binding module or a heterologous prepro sequence.
 21. An isolated, synthetic or recombinant polypeptide having an amino acid sequence of the polypeptide of claim 3, and further comprising a heterologous signal sequence or a heterologous prepro sequence.
 22. The isolated, synthetic or recombinant polypeptide of claim 1, wherein the xylanase activity comprises catalyzing hydrolysis of a polysaccharide.
 23. An isolated, synthetic or recombinant polypeptide having a xylanase activity comprising a polypeptide encoded by a nucleic acid capable of hybridizing under stringent conditions to the complement of the sequence of SEQ ID NO:159, wherein the stringent conditions comprise a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes.
 24. A composition comprising the isolated, synthetic or recombinant polypeptide of claim
 1. 25. The composition of claim 24 formulated in a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel form, a paste or a slurry form.
 26. The composition of claim 24 formulated as a wood, a wood product, a wood pulp, a Kraft pulp, a paper, a paper product, a paper pulp, or a combination thereof.
 27. The composition of claim 26, wherein the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp or combination thereof comprises a softwood and/or a hardwood, or the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp, or combination thereof, is derived from a softwood and/or a hardwood.
 28. The composition of claim 24 comprising ethanol or an alcohol.
 29. The composition of claim 24 formulated as a dough or a bread product.
 30. The composition of claim 24 formulated as a food, a food supplement, a feed, a feed supplement, or a nutritional supplement.
 31. The composition of claim 24 formulated as a detergent composition.
 32. A method of producing a recombinant polypeptide having the sequence of claim 1 comprising the steps of: (a) providing a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises a sequence encoding the polypeptide of claim 1; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide.
 33. A method for hydrolyzing, breaking up or disrupting a polysaccharide-, a cellulose-, a hemicellulose- or xylan-comprising composition comprising the following steps: (a) providing the polypeptide having a xylanase activity of claim 1, or the composition of claim 24; (b) providing a composition comprising a polysaccharide, a cellulose, a hemicellulose or a xylan; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the xylanase hydrolyzes, breaks up or disrupts the polysaccharide-, cellulose-, hemicellulose- or xylan-comprising composition.
 34. A method for hydrolyzing, breaking up or disrupting a polysaccharide-, a cellulose-, a hemicellulose- or xylan-comprising composition comprising contacting the polysaccharide-, a cellulose-, a hemicellulose- or xylan-comprising composition with a polypeptide having a xylanase activity comprising the polypeptide of claim
 1. 35. A method for reducing or releasing lignin from a wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp, or combination thereof, comprising contacting the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp or combination thereof, with the polypeptide of claim 1 or the composition of claim
 24. 36. A method for reducing or releasing a lignin from a lignin-comprising composition, comprising contacting the lignin-comprising composition with the polypeptide of claim
 1. 37. A method for treating a wood, a wood product, a wood pulp, a Kraft pulp, a paper, a paper product, a paper pulp, or a combination thereof, comprising contacting the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp or a combination thereof, with the polypeptide of claim 1 or the composition of claim
 24. 38. The method of claim 37, wherein the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp or combination thereof comprises a softwood and/or a hardwood, or the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp or combination thereof, is derived from a softwood and/or a hardwood.
 39. A method for bleaching or decoloring a wood, a wood product, a wood pulp, a Kraft pulp, a paper, a paper product, a paper pulp, or a combination thereof, comprising contacting the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp, or combination thereof with the polypeptide of claim 1 or the composition of claim
 24. 40. A method for bleaching or decoloring a composition comprising contacting the composition with the polypeptide of claim
 1. 41. A method for reducing the use of bleaching chemicals in a wood, a wood product, a wood pulp, a Kraft pulp, a paper, a paper product, a paper pulp, or a combination thereof bleaching process comprising biobleaching the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp, or combination thereof with the polypeptide of claim 1 or the composition of claim
 24. 42. The method of claim 41, wherein said bleaching chemical comprises chlorine, chlorine dioxide, a peroxide, or any combination thereof.
 43. A method for producing a fermentable sugar comprising: (a) providing at least one polypeptide having a xylanase activity comprising the amino acid sequence of claim 1; (b) providing a composition comprising a polysaccharide, a xylan, a hemicellulose or a cellulose; and (c) contacting the composition of step (b) with the polypeptide of claim 1, thereby enzymatically hydrolyzing the polysaccharide, xylan, hemicellulose or cellulose to produce a fermentable sugar.
 44. The method for producing a fermentable sugar of claim 43 wherein the composition comprising a polysaccharide, xylan, a hemicellulose or a cellulose is a wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp, or combination thereof, or comprises a softwood and/or a hardwood, or the wood, wood product, wood pulp, Kraft pulp, paper, paper product, paper pulp, or combination thereof is derived from a softwood and/or a hardwood.
 45. A method for making a food, a food supplement, a feed, a feed supplement or a nutritional supplement comprising adding the polypeptide of claim 1 with or to the food, food supplement, feed, feed supplement, or nutritional supplement. 